CDDO-compounds and combination therapies thereof

ABSTRACT

CDDO-compounds in combination with other chemotherapeutic agents induce and potentiate cytotoxicity and apoptosis in cancer cell. One class of chemotherapeutic agents include retinoids. Cancer therapies based on these combination therapies are provided. Also provided are methods to treat graft versus host diseases using the CDDO compounds.

BACKGROUND OF THE INVENTION

[0001] The present application is a continuation-in-part of co-pendingU.S. Patent Application Serial No. 60/253,673 filed Nov. 28, 2000. Theentire text of each of the above-referenced disclosures is specificallyincorporated by reference herein without disclaimer.

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the fields of cancertherapy. More particularly, it concerns the use of triterpenoidCDDO-compounds, such as CDDO and/or methyl-CDDO, in combination withother chemotherapeutic agents for the treatment of cancers.

[0004] 2. Description of Related Art

[0005] Cancer has become one of the leading causes of death in thewestern world, second only behind heart disease. Current estimatesproject that one person in three in the U.S. will develop cancer, andthat one person in five will die from cancer. Major challenges remain tobe overcome for all cancers, but this is especially true for thehematological malignancies. For example, increasing the cure rate foracute lymphoblastic leukemia (ALL), especially for middle-aged and olderadults has proved difficult. Despite high rates of complete remission,many patients relapse after chemotherapy. Chronic lymphocytic leukemia(CLL), while slow-progressing and well responding initially, frequentlytransforms into a drug-resistant disease.

[0006] Therapeutic regimens employed in the therapy of acute myelogenousleukemia (AML) have not changed over the last three decades and usuallyencompass ara-C and anthracycline analogs (Andreef, 1995). Use of newdrugs such as topoisomerase inhibitors, cytokines and MDR-1 blockershave failed to impact AML patient survival (Kornblau et al., 1997;Greenberg et al., 1999; Kolitz et al., 1999; Estey et al., 1998; Beranet al., 1999). The recently synthesized new and unique triterpenoid,CDDO, has anti-proliferative effects in many human tumor cell lines(Suh, 1999), induces apoptosis in non-small cell lung cancer cells (Kimet al., 2000) and has anti-proliferative and pro-apoptotic properties inseveral leukemias (Konopleva et al., 1999a).

[0007] Recently, the knowledge of mechanisms controlling apoptoticpathways has increased. In general, multiple signaling pathways leadfrom death-triggering extracellular or intracellular signals to acentral control followed by an execution stage. At this stage,CED3/caspases are activated, leading to the characteristic apoptoticstructural lesions accompanying cell death which include cytoplasmic andchromatin condensation and DNA fragmentation. Two regulatory pathwayshave been elucidated. The death receptor pathway (also called the“extrinsic” pathway), which is triggered by members of the tumornecrosis family (TNF) family, and is mediated by recruitment of theproximal regulator caspase 8 to the death receptor complex. Theactivated initiator caspases in turn activate the effector caspases 3, 6and 7. The other pathway (called the “intrinsic” pathway) involves themitochondria and is regulated by the Bcl-2 family of proteins. In thispathway, mitochondrial sequestration or release of cytochrome C (Yang etal., 1997) is followed by the activation of Apaf-1, caspase 9, andcaspase 3 (for review, see (Konopleva and Andreeff, 1999; Konopleva etal., 1998; Kornblau et al., 1999).

[0008] Most chemotherapeutic agents used in the treatment ofhematological malignancies cause cell killing by inducing apoptosis.Newer approaches attempt to induce apoptosis by directly targetingapoptotic pathways. For example, agents that trigger the signaling ofFas or TRAIL receptors induce the extrinsic pathway at the cell surface.Activation of the retinoic acid receptors also results in apoptosis ordifferentiation via down-modulation of Bcl-2 and Bcl-X_(L) mRNA andprotein levels (Andreef et al., 1999; Agarwal and Mehta, 1997). Clinicaltrials of several of these agents are under way. The most strikingimprovement in AML therapy came with the introduction ofall-trans-retinoic acid (ATRA) for the treatment of acute promyelocyticleukemia (APL) (Castaigne et al., 1990). Early mortality of APLdecreased, and over 90% of patients achieved complete remissions(Warrell et al., 1991) including some molecular remissions with PCRnegativity (Estey et al., 1999). The peroxisome proliferator-activatedreceptor (PPAR) is a member of nuclear receptor family that is involvedin apoptosis. Mutations of PPAR gene products are seen in several cancertypes demonstrating the role of PPAR in cancer. Thus, PPAR based cancertherapies are another approach for anticancer chemotherapeutics.

[0009] Although some agents that target particular points of apoptoticpathways have anti-leukemic activities, none have proven optimal fortreatment. There is still a need to systematically investigate newagents and to provide treatment regimens for hematological and othercancers.

SUMMARY OF THE INVENTION

[0010] The present invention overcomes deficiencies in the art andprovides an anti-cancer therapy that involves the combination ofCDDO-compounds, such as CDDO and methyl-CDDO, with other conventionalchemotherapeutic compounds and/or with chemotherapeutic agents thatactivate different parts of apoptotic cascades.

[0011] Therefore, provided in the invention is a method for inducingcytotoxicity in a cell comprising contacting the cell with aCDDO-compound and a chemotherapeutic agent, wherein the combination ofthe CDDO-compound with the chemotherapeutic agent is effective ininducing cytotoxicity in the cell. The CDDO-compound is CDDO ormethyl-CDDO.

[0012] In one embodiment of the method, the CDDO-compound is contactedwith the cell prior to contacting the cell with the chemotherapeuticagent. In another embodiment of the method, the chemotherapeutic agentis contacted with the cell prior to contacting the cell with CDDO.

[0013] In other embodiments of the method, the cell is a cancer cell. Insome aspects the cancer cell is a leukemic cell. In more specificaspects, the leukemic cell is a blood cancer cell, a myeloid leukemiacell, a monocytic leukemia cell, a myelocytic leukemia cell, apromyelocytic leukemia cell, a myeloblastic leukemia cell, a lymphocyticleukemia cell, an acute myelogenous leukemic cell, a chronic myelogenousleukemic cell, a lymphoblastic leukemia cell, a hairy cell leukemiacell.

[0014] In yet other embodiments, the cancer cell is a solid tumor cell.In specific aspects, the solid tumor cell is a bladder cancer cell, abreast cancer cell, a lung cancer cell, a colon cancer cell, a prostatecancer cell, a liver cancer cell, a pancreatic cancer cell, a stomachcancer cell, a testicular cancer cell, a brain cancer cell, an ovariancancer cell, a lymphatic cancer cell, a skin cancer cell, a brain cancercell, a bone cancer cell, a soft tissue cancer cell.

[0015] In one embodiment of the method, the cell is located in a humansubject. In one embodiment, the CDDO-compound may be administeredlocally. Therefore, the compound may be administered by intratumoralinjection and/or by injection into tumor vasculature.

[0016] In another embodiment of the method, the CDDO-compound may beadministered systemically. In other specific aspects of this embodiment,the CDDO-compounds may be administered intravenously, intra-arterially,intra-peritoneally, orally, and/or during ex vivo bone marrow or bloodstem cell purging. CDDO may be administered at dosages in the range of5-30 mg/kg intravenously (i.v.) or 5-100 mg/kg orally. Thus, about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or about 100 mg/kg of CDDO may be administered by i.v. or may beadministered orally. CDDO-Me may be administered in the range of 5-100mg/kg intravenously or 5-100 mg/kg orally for 3-30 days. Thus, about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,or 100 mg/kg of CDDO may be administered by i.v. or, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100mg/kg of CDDO may be administered orally. The skilled artisan willappreciate that these dosages are only guidelines and a physician willdetermine exact dosages at the time of administration factoring in otherconditions such as age, sex, disease, etc. of the patient.

[0017] In one embodiment, the chemotherapeutic agent may be one or moreof the listed chemotherapeutics including, doxorubicin, daunorubicin,dactinomycin, decitabine, mitoxantrone, cisplatin, procarbazine,mitomycin, carboplatin, bleomycin, etoposide, teniposide,mechlroethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil,ifosfamide, melphalan, hexamethylmelamine, thiopeta, busulfan,carmustine, lomustine, semustine, streptozocin, dacarbazine, adriamycin,5-fluorouracil (5FU), camptothecin, actinomycin-D, hydrogen peroxide,nitrosurea, plicomycin, TRAIL, tamoxifen, taxol, transplatinum,vincristin, vinblastin, mylotarg, dolastatin-10, bryostatin andmethotrexate. However, one of ordinary skill in the art will appreciatethat the invention is not limited to these chemotherapeutic agents andmay involve the use of other DNA damaging agents as well.

[0018] It is also contemplated that the chemotherapeutic agent can be anagent that causes immunosupression and may be a corticosteroid ortacrolimus (also known as SK506). Especially in embodiments that concernex vivo bone marrow or blood cell purging, the bone marrow or blood maybe treated with a CDDO compound, either alone or in conjunction with anyother agent to eliminate any tumor, malignant or leukemic cell beforetreating the patient.

[0019] In yet other embodiments, the chemotherapeutic agent is aretinoid. The retinoid may be all-trans-retinoic acid (ATRA),9-cis-retinoic acid, LG100268, LGD1069 (Targretin, bexarotene),fenretinide [N-(4-hydroxyphenyl)retinamide, 4-HPR], CD437 or any RXR- orRAR-specific retinoic acid. In one specific embodiment, the RXR-specificretinoic acid is LG100268 (Ligand Pharmaceuticals). In some embodiments,the retionids may be administered as liposomal formulations. Theseliposomal formulations may be administered intravenously or throughother routes as well, for example a liposomal formulation of ATRA isadministered a range of 10-100 mg/m²/day intravenously. Thus, one mayadminister 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100 mg/m²/day of a liposomal formulation of ATRA. In onespecific embodiment, 90 mg/m²/day of ATRA as a liposomal formulation isintravenously. In other embodiments, the retinoids may be administeredorally. For example, ATRA may be administered in the range of 10-100mg/m²/day. Thus, one may administer 10, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/m²/day of ATRA. In onespecific embodiment, ATRA may be administered at 45 mg/m²/day orallydaily. In another example, 9-cis-Retinoid acid may be administered inthe range of 20-150 mg/m² twice a day orally. Thus, one may administer20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 mg/m² of9-cis-retinoid. LG100268 may be effective in a dose range of 5-50 mg/kg.Thus, 5, 10, 15, 20, 25, 30, 35, 40, 45, to 50 mg/kg of LG100268 may beadministered. LGD1069 (Targretin, bexarotene) capsules are contemplatedfor the topical treatment of cutaneous lesions in patients withcutaneous T-cell lymphoma (CTCL) who have refractory or resistantdisease after other therapies. The dose ranges of these capusles is300-400 mg/m²/day orally. Thus, 300, 350, 400 mg/m²/day may be used.LGD1069 gel at 1% may also be used for the topical treatment ofcutaneous lesions in patients with CTCL (Stage (1A and 1B) who haverefractory or resistant disease after other therapies; two to four timesdaily. Fenretinide [N-(4-hydroxyphenyl)retinamide, 4-HPR] iscontemplated useful at 25-600 mg daily and the administration in someembodiments may be continuous. Thus, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540,560, 580, 600 mg may be administered daily. Of course, the skilledartisan will understand that while these dosage ranges provide usefulguidelines appropriate adjustments in the dosage depending on the needsof an individual patient factoring in disease, gender, age and othergeneral health conditions will be made at the time of administration toa patient by a trained physician.

[0020] In some embodiments of the method, the cell is contacted with theCDDO-compound a second time. In yet other embodiments, the cell may becontacted with the chemotherapeutic agent a second time. In still otheraspects of this method, the CDDO-compound and the chemotherapeutic agentcan be contacted with the cell at the same time.

[0021] One embodiment of the method, further comprising tumor resectionin conjunction with the CDDO-compound based combination therapy. Thetumor resection may occurs prior to the contacting. Thus, the contactingcan comprises treating a resected tumor bed with the CDDO-compound andthe chemotherapeutic agent. In other aspects, the tumor resection occursafter the contacting. In still other aspects, the contacting occurs bothbefore and after the tumor resection.

[0022] The invention also provides methods of killing a tumor cellcomprising contacting the tumor cell with a CDDO-compound and achemotherapeutic agent, wherein the combination of said CDDO-compoundwith said chemotherapeutic agent, induces killing of said tumor cell.

[0023] The invention also provides methods of inducing apoptosis in atumor cell comprising contacting said tumor cell with a CDDO-compoundand a chemotherapeutic agent, wherein the combination of saidCDDO-compound with said chemotherapeutic agent, induces apoptosis ofsaid tumor cell. The CDDO-compound is CDDO or methyl-CDDO. In someembodiments of this method, the chemotherapeutic agent is a retinoid.

[0024] Also provided are methods for inducing differentiation in a tumorcell comprising contacting the tumor cell with a CDDO-compound and achemotherapeutic agent, wherein the combination of the CDDO-compoundwith the chemotherapeutic agent, induces the differentiation of thetumor cell.

[0025] Further provided are methods for treating cancer in a humanpatient comprising administering a CDDO-compound and a chemotherapeuticagent to the human patient, wherein the combination of the CDDO-compoundwith the chemotherapeutic agent, is effective to treat the cancer.

[0026] The invention also describes methods of potentiating the effectof a chemotherapeutic agent on a tumor cell comprising contacting thetumor cell with a CDDO-compound and the chemotherapeutic agent.

[0027] In addition, the invention provides methods of inhibiting growthof a tumor cell comprising contacting the tumor cell with aCDDO-compound and a chemotherapeutic agent.

[0028] In all these methods, the CDDO-compound can be CDDO (2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid) or methyl-CDDO. In some embodiments,the chemotherapeutic agent is a retinoid. In some specific aspects, theretinoids are all-trans-retinoic acid (ATRA), 9-cis-retinoic acid,LG100268, LGD1069 (Targretin, bexarotene), fenretinide[N-(4-hydroxyphenyl)retinamide, 4-HPR], CD437 or any RXR- orRAR-specific retinoic acid. In additional embodiments, otherchemotherapeutics described above and elsewhere in the specification mayalso be used.

[0029] In other embodiments, the invention provides methods for thetreatment and prevention of graft versus host disease (GVHD) byproviding a CDDO-compound either alone or in conjunction with anotheragent, such as an immunosupressive agent or a chemotherapeutic agent forthe treatment of GVHD. In graft versus host disease the donor immunesystem mounts a response against the host's organs or tissue. As CDDOcompounds, either alone or in conjunction with other agents, can induceapoptosis by inhibiting Bcl-2 and have activity in lymphoid tissue, theinventors contemplate that CDDO-compound based therapies can be used toprovide therapy for graft versus host diseases.

[0030] Thus, the invention provides methods of inducing apoptosis in alymphoid cell that expresses Bcl-2 comprising contacting said lymphoidcell with a CDDO-compound and an immunosupressive agent. The Bcl-2 maybe expressed either endogenously or exogenously. In the case ofexogenous Bcl-2 expression the Bcl-2 is expressed by a expression vectorthat comprises a nucleic acid that encodes Bcl-2 under the control of apromoter active in the lymphoid cell. Methods for achieving exogenousexpression of nucleic acids are well known in the art and are describedelsewhere in the specification. Such methods are also described inSambrook and Maniatis (1993), incorporated herein by reference.

[0031] In some embodiments, the lymphoid cell is a T-cell. In otherembodiments, the lymphoid cell is a cancer cell. In yet otherembodiments, the lymphoid cell is located in a human. Although anyimmunosupressive agent known in the art can be used some non-limityingexamples include corticosteroids and/or tacrolimus (SK506).

[0032] In some embodiments, the lymphoid cell is further additionallycontacted with a chemotherapeutic agent.

[0033] The invention also provides methods of treating or preventinggraft versus host disease in a subject comprising administering to thesubject a CDDO-compound in combination with an immunosupressive agent.In some embodiments, the subject is further treated with achemotherapeutic agent. The CDDO-compound is CDDO or methyl-CDDO.

[0034] In some embodiments, the subject is a human. In otherembodiments, the subject has cancer. In yet other embodiments, thesubject has received autologus bone marrow transplantation.

[0035] In some aspects the CDDO-compound is administered during ex vivopurging. Treatment of bone marrow or blood stem cells withCDDO-compounds, alone or with other agents, eliminates any tumor cellsor leukemic cells or malignant cells. In other aspects the CDDO-compoundis administered locally, for example, by direct intratumoral injectionor by injection into tumor vasculature. In yet other aspects, theCDDO-compound is administered systemically, for example, intravenously,intra-arterially, intra-peritoneally, or orally.

[0036] Following longstanding patent law convention, the word “a” and“an”, when used in conjunction with the word comprising, mean “one ormore” in this specification, including the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The following drawings form part of the present specification andare included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

[0038]FIG. 1. CDDO decreases cell number in HL-60 cells.

[0039]FIG. 2. CDDO inhibits proliferation and induces apoptosis.

[0040]FIG. 3. CDDO induces apoptosis in myeloid cell lines.

[0041]FIG. 4. CDDO enhances ara-C cytotoxicity in HL60-DOX cells.

[0042]FIG. 5. CDDO alone and in combination with ara-C induces apoptosisin primary AML cells.

[0043]FIG. 6. CDDO combined with ara-C induces differentiation inprimary AML cells.

[0044]FIG. 7. CDDO decreases colony formation of AML blasts.

[0045]FIG. 8. CDDO induces decreases in mitochondrial potential(CMXRos). Annexin V-positivity and Caspase Cleavage.

[0046]FIG. 9. CDDO decreases Bcl-2 m-RNA in HL-60 cells.

[0047]FIG. 10. CDDO decreases XIAP m-RNA in HL-60 cells.

[0048]FIG. 11A. & FIG. 11B. FIG. 11A. Binding of [3H]-rosiglitazone toPPARγ (cold CDDO as competitor). FIG. 11B. CDDO transactivates PPARγ.

[0049]FIG. 12A. & FIG. 12B. PPARγ protein expression (western blot).

[0050]FIG. 13A. & FIG. 13B. PPARγ is expressed in myeloid cell lines andprimary AML samples.

[0051]FIG. 14. ATRA enhances CDDO-induced growth inhibition of HL60cells (72 hrs).

[0052]FIG. 15. ATRA (1 μM) enhances CDDO-induced cytotoxicity inleukemic cell lines.

[0053]FIG. 16. ATRA enhances CDDO-induced differentiation of HL60 cells(72 hrs).

[0054]FIG. 17. CDDO combined with ATRA decreases Bcl-2 mRNA in U937cells (24 hrs).

[0055]FIG. 18A. & FIG. 18B. FIG. 18A. LG-100268 synergistically enhancesCDDO-induces killing in HL-60 cells. FIG. 18B. LG-100268 increasesCDDO-induces killing in HL-60/RXR cells but not in HL-60/RAR cells.

[0056]FIG. 19. Southern blot, NOD/Scid BM.

[0057]FIG. 20A. and FIG. 20B. CDDO-Me inhibits cell growth and inducesapoptosis in HL-60 cells.

[0058]FIG. 21A. and FIG. 21B. CDDO-Me induces apoptosis in primary AMLsamples.

[0059]FIG. 22. CDDO-Me inhibits AML clonogenic progenitor growth.

[0060]FIG. 23. CDDO-Me induces Annexin V positivity in leukemic cells.

[0061]FIG. 24. CDDO-Me induces caspase activation and decreasesmitochondrial potential in U937 cells.

[0062]FIG. 25A. and FIG. 25B. Caspase-3 inhibitor DEVD blocks CDDO-Meinduced annexin V positivity and caspase-3 cleavage.

[0063]FIG. 26. CyA partially inhibits CDDO-Me-induced loss ofmitochondrial potential.

[0064]FIG. 27A, FIG. 27B and FIG. 27C. CDDO-Me induces Bax expressionand caspase-3 cleavage.

[0065]FIG. 28A and FIG. 28B. Overexpression of Bcl-2 and Bcl-Xl inhibitsCDDO-Me induced apoptosis in HL-60 cells.

[0066]FIG. 29. Caspase-8 inhibitor IEDT prevents CDDO-Me inducedapoptosis in NB4 cells.

[0067]FIG. 30A and FIG. 30B. ATRA enhances CDDO-Me-induced cytotoxicityin HL-60 cells.

[0068]FIG. 31. ATRA (1 μM) enhances CDDO-Me-induced cytotoxicity inleukemic cell lines.

[0069]FIG. 32. ATRA enhances CDDO-Me-induced apoptosis in primary AMLsample.

[0070]FIG. 33. cDNA array: CDDO-Me decreases VEGFR1 expression in U937cells.

[0071]FIG. 34. CDDO induces histone acetylation in HL-60/RXR cells.

[0072]FIG. 35. Activity of CDDO against breast-cancer cells in vivo.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0073] Therapeutic regimens employed in the therapy of hematologicalmalignancies have not proved very effective. For example, the cure ratefor acute lymphoblastic leukemia (ALL), is very low and patients oftensuffer relapses after chemotherapy. In the case of chronic lymphocyticleukemia (CLL), the disease often transforms into a drug-resistantdisease.

[0074] The present invention provides methods for anticancer therapythat comprise administering triterpenoid CDDO-compounds in combinationwith other chemotherapeutic agents. The term “CDDO-compounds” used inthis specification refers to CDDO and methyl-CDDO (CDDO-Me). Among thetwo CDDO-compounds, CDDO-Me was found to be more potent.

[0075] The present invention demonstrates that CDDO-compounds incombination with a chemotherapeutic compound are very effective ininhibiting tumor cell growth; inducing apoptosis; inducingdifferentiation; and/or inducing cell death in several leukemic cellsand cell lines. In addition, the induction of apoptosis by a combinationof the CDDO-compound and a chempotherapeutic agent permits effectivetreatment using much lower doses of both agents as compared to thedosage of either agent alone.

[0076] Some of the chemotherapeutic agents, that are used in combinationwith the CDDO-compounds, target specific apoptotic pathways. One suchclass of chemotherapeutics are the retinoids. Therefore, the inventionprovides the use of retinoids, especially the RXR-specific ligands, asthe other chemotherapeutic agents in combination with the CDDO-compoundsas highly effective cancer treatments. CDDO-compounds are ligands ofPPARγ receptors which form heterodimers with retinoid receptors. Thus,the anticancer properties of the CDDO-compounds are potentiated whencombined with retinoids.

[0077] In addition, chemotherapeutic agents that may be used incombination with the CDDO-compounds include all standard and commonlyused chemotherapeutic agents known in the art. Examples of these aredescribed ahead in the specification and include DNA-damaging agentssuch as, ara-C, doxorubicin, danorubicin etc.

[0078] The invention also contemplates the use of other PPARγ ligands aschemotherapeutic agents. Also contemplated are the use ofimmunosuppressive agents such as corticosteroids and tacrolimus.

[0079] The present invention demonstrates an increase in cancer celldestruction compared to surrounding normal tissue and indicates thatCDDO-compounds when combined with chemotherapy, provide a clinicallyuseful tool.

[0080] As CDDO-compounds inhibit Bcl-2 and are effective in lymphoidcells, another aspect the invention concerns the treatment of graftversus host disease (GVHD) by providing a CDDO-compound either alone orin conjunction with another agent, such as an immunosupressive agent ora chemotherapeutic agent for the treatment of GVHD.

[0081] A. CDDO-Compounds

[0082] CDDO. CDDO (2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid) is anovel synthetic triterpenoid with potent differentiating,anti-proliferative and anti-inflammatory activity and was synthesizedpreviously by the inventors (Suh et al., 1999). It inhibitsproliferation of different tumor cell lines and induces monocyticdifferentiation of myeloid U937 cells. Recently, CDDO was found to be aspecific ligand for PPARγ, while transactivation assays withglucocorticoid, estrogen, progesteron, and retinoid receptors werenegative (Suh et al., 1999). The inventors have demonstrated that CDDOexerts strong antiproliferative, and apoptotic effects on leukemic celllines and primary AML in vitro and also induces monocyticdifferentiation of leukemic cell lines and some primary AMLs. CDDO alsomediated reduction in colony formation in AML progenitors as comparedwith normal CD34⁺ cells (Konopleva et al., 1999). This differentialeffect on normal vs. leukemic progenitor cells is useful for AMLtherapy. The present invention shows that this effect is profoundlyincreased by combination of CDDO with retinoids such as all-transretinoic acid (ATRA) in HL-60 cells. CDDO combined with ATRA alsoexhibits an enhanced pro-apoptotic effect. In addition to ATRA, otherretinoids contemplated as useful include 9-cis retinoic acid, LG100268,LGD1069 (Targretin, bexarotene), fenretinide[N-(4-hydroxyphenyl)retinamide, 4-HPR] and CD437. Furthermore, theinvention shows that CDDO-compounds increases ara-C cytotoxicity. Theinvention also shows that CDDO in combination with otherchemotherapeutic agents has potent anticancer effects.

[0083] Method of Synthesis of CDDO.

[0084] CDDO may be synthesized by the scheme outlined below.

[0085] Methyl-CDDO. Methyl-CDDO (CDDO-Me), the C-28 methyl ester ofCDDO, also exerts strong antiproliferative and apoptotic effects onleukemic cell lines and in primary AML samples in vitro as well asinduces monocytic differentiation of leukemic cell lines and someprimary AMLs. Thus, CDDO-Me provides chemotherapy for the treatment ofleukemias. The present invention demonstrates that this effect isprofoundly increased by combination of CDDO-Me with otherchemotherapeutic agents. These include retinoids such as ATRA, 9-cisretinoic acid, , LG100268, LGD1069 (Targretin, bexarotene), fenretinide[N-(4-hydroxyphenyl)retinamide, 4-BPR], CD437 and other RXR andRAR-specific ligands. This combination also increases ara-Ccytotoxicity, further reduces AML colony formation, inhibits ERKphosphorylation and promotes Bcl-2 dephosphorylation, and inhibits invitro angiogenesis. The ability of CDDO-Me in combination with retinoidsto induce differentiation in leukemic cells in vitro show that thesecompounds may have similar in vivo effects. The anti-angiogenicproperties of CDDO-Me further increase its potent anti-leukemia activityin combination with retinoids. Furthermore, CDDO-Me was found to be morepotent at lower concentrations than CDDO.

[0086] Method of Synthesis of CDDO-Me.

[0087] CDDO-Me may be synthesized by the scheme outlined below.

[0088] The present invention provides combinations of CDDO-compounds andchemotherapeutic agents that are useful as treatments for cancers andhematological malignancies. In one embodiment, the chemotherapeutics areretinoids. As CDDO-compounds are PPARγ ligands and PPARγ is known to bealtered in many types of cancers, the inventors contemplate, thatligation of PPARγ in combination with retinoids such as, RXR-specificligands, provides a mechanistic basis for maximal increase intranscriptional activity of the target genes that control apoptosis anddifferentiation. The CDDO-compounds and retinoids in combinationdemonstrate an increased ability to induce differentiation, inducecytotoxicity, induce apoptosis, induce cell killing, reduce colonyformation and inhibit the growth of several types of leukemic cells.

[0089] B. Retinoids

[0090] Retinoids are a group vitamin A derivatives that have potentialapplication in chemoprevention and in therapy of many types ofmalignancies. They have been used as chemotherapeutics for the treatmentand prevention of a variety of cancerous and pre-cancerous conditions,such as melanoma, cervical cancer, some forms of leukemia, oralleukoplakia and basal and squamous cell carcinomas. Retinoids can alsomodulate programmed cell death (apoptosis) and are therefore importantto cancer therapy. They act via interaction with two major classes ofnuclear receptors, the retinoid acid receptors (RARs) and the retinoid Xreceptors (RXRs) that function as dimeric, ligand-dependenttranscription factors (Pfahl et al., 1994). The apoptosis controllingprotein, Bcl-2, functions as a downstream regulator of retinoid-inducedcell growth and differentiation in hematopoietic cells.

[0091] All-trans-retinoic acid (ATRA) belongs to the retinoid family ofligands for nuclear receptors. Other retinoid ligands used hereininclude 9-cis-retinoic acid, LG-100286, Fenretinide[N-(4-hydroxyphenyl)retinamide, 4-HPR], LG1069 (also alternatively knownas Targretin, bexarotene) and CD-437 are some other retinoidscontemplated. CD437 is a novel retinoid that binds to both RARγ and RARβretinoids receptors. It is a potent inducer of apoptosis in vitro. Notrials in humans have been conducted. In mice, oral administration of10-30 mg/kg daily for 3 wk or injection of 10 mg/kg of body weight inthe tumor caused growth inhibition of melanoma xenografts in vivo(Schadendorf, et al., 1996). However, one of skill in the art willrecognize that one may use any other retinoid. Such a retinoid may benaturally occurring or synthetic. The retinoid may further be a ligandof the RXR receptors and/or the RAR receptors.

[0092] Retinoids may be administered by any route as described hereinand in other parts of this specification. In some specific embodiments,ATRA may be administered at a range of 10-100 mg/m²/day, for example, at45 mg/m²/day orally daily. A liposomal formulation of ATRA may beadministered at 90 mg/m²/day IV. 9-cis-Retinoid acid may be administeredat a range of 20-150 mg/m² twice a day orally. LG100268 in mice modelswas administered at a dose of 5-10 mg/kg. LGD1069 is contemplated asuseful for the topical treatment of cutaneous lesions in patients withcutaneous T-cell lymphoma (CTCL) who have refractory or resistantdisease after other therapies. In some embodiments the LGD1069 isadministered as capsules of 300-400 mg/m²/day taken orally. In otherembodiments, LGD1069 is administered as a gel of about 1% strength forthe topical treatment of cutaneous lesions in patients with CTCL (Stage1A and 1B) who have refractory or resistant disease after other cancertherapies and may be taken two to four times daily. Fenretinide[N-(4-hydroxyphenyl)retinamide, 4-HPR] is contemplated useful at 25-600mg daily and may be administered continuously in some embodiments. Oneof skill in the art will understand that while these dosage rangesprovide useful guidelines appropriate adjustments in the dosagedepending on the needs of an individual patient factoring in disease,gender, age and other general health conditions will be made by thetrained physician.

[0093] C. PPAR

[0094] The peroxisome proliferator-activated receptor (PPAR) is a memberof a nuclear receptor family that is involved in apoptosis. This familyincludes receptors for the steroid, thyroid and retinoid hormones thatoften serve as transcription factors. The three known human PPARsubtypes, α, γ and δ, show distinct tissue distribution and areassociated with selective ligands (Forman et al., 1997; Kliewer et al.,1994; Wilson et al., 1996). PPARγ is expressed at high levels in adiposetissue and in macrophages and can induce cell cycle arrest anddifferentiation of preadipocyte cells. As these receptors regulate keygenes involved in cellular homeostasis and differentiation, they havevalue as therapeutic targets.

[0095] The present inventors show that the CDDO-compounds are PPARγligands. Endogenous PPARγ ligands include fatty acid-like compounds suchas 15-deoxyΔ^(12,14)PGJ2 and linoleic acid (Forman et al., 1995; Klieweret al., 1995; Nagy et al., 1998). Some examples of PPAR pharmaceuticalligands include the thiazolidinediones (TZDS) such as troglitazone,BRL49653 (rosiglitazone), and pioglitazone, and non-steroidalanti-inflammatory drugs (Lehmann et al., 1997), L-805645, GW347845X.TZDs are used for the treatment of type 2 diabetes because theysensitize tissues to insulin (Lehmann et al., 1995).

[0096] PPARγ shares structural similarities with other nuclear-receptorfamily members, including a central DNA-binding domain and acarboxy-terminal ligand-binding domain (LBD). All nuclear receptorsrequire the transcription activation function (AF-2) domain that islocated in the C-terminus of the LBD (Evans, 1988), for the recruitmentof the coactivator SRC-1. PPARγ must form a heterodimer with RXR to bindDNA and activate transcription (Nolte et al., 1998). PPAR-RXRheterodimers can be activated by PPAR or RXR ligands (Forman et al.,1997), and RXR-specific ligands markedly induce the binding of SRC-1 toPPARγ-RXR heterodimers (Westin et al., 1998). Assembly of this complexresults in a large increase in transcriptional activity. The presentinventors contemplate that one could increase the effects of PPARγligands by combining them with ligands specific for RXR. For example,the present inventors have shown that combination of CDDO with aRXR-specific ligand, such as ATRA, decreases cell viability and inducesterminal differentiation in myeloid leukemic cell lines. InRXR-expressing HL-60 cells CDDO in combination with a RXR ligand induceshistone acetylation. Additionally, the inventors tested the effects ofthe PPARγ ligands listed above alone and in combination with retinoidsand found an increased differentiation followed by apoptosis in leukemiccells.

[0097] It has also been shown that simultaneous activation of bothreceptors can yield maximal antidiabetic activation in vivo (Mukherjeeet al., 1997). For example, combinations of the PPARγ ligands, 15D-PGJ2or BRL49563 with RXR-specific ligand LG100268 markedly decreased cellgrowth and induced monocytic differentiation in HL-60 cells indicatingthat activation of the PPARγ/RXR heterodimer represents a novelregulatory pathway for HL-60 maturation (Tontonoz et al., 1998). As SRCco-activator proteins possess intrinsic histone acetyltransferaseactivity, ligand-mediated receptor transactivation may involve targetedhistone acetylation of chromatin by recruited coactivators.

[0098] Transactivation of PPARγ target genes is a multi-step processthat first involves binding of the PPARγ/RXR heterodimer to specificDR1-type response elements in the promoter of a target gene. In theabsence of ligand, this heterodimer associates with a complex ofco-repressor proteins that silence the promoter by deacetylatinghistones in the adjacent chromatin. Ligand binding induces aconformational change in the receptor, which dissociates theco-repressor complex, and permits the heterodimer to interact with atleast two co-activator complexes, namely p160/CBP and DRIP (also calledTRAP or ARC)-FIG. 1. These two complexes acetylate histones (makingadjacent chromatin more accessible) and bridge the PPARγ/RXR heterodimerto the basal transcriptional machinery, thus driving transcription ofthe target gene. This ligand-induced transactivation is dependent on: 1)the different types of co-repressors and co-activators associated withthe receptor heterodimer, and 2) the relative affinities of thesecofactors for PPARγ and RXR. Indeed, it has already been shown that RXRand PPARγ agonists recruit different co-activators to the heterodimer(Yang et al., 2000).

[0099] There is emerging evidence of the involvement of PPAR-signalingin cancer as shown by the following: 1. High expression of PPARγ mRNAand protein has been observed in six colon cancer cell lines (Kitamuraet al., 1999). Troglitazone, a selective PPARγ ligand, causes markedcell cycle arrest and enterocyte differentiation markers. In addition,four somatic PPARγ mutations were found among 55 sporadic colon cancers.Each greatly impaired the function of the protein (Sarraf et al., 1999).These data demonstrate that colon cancer in humans is associated withloss-of-function mutations in the PPARγ. 2. Significant PPARγ expressionis detected in most human metastatic breast cancers (Mueller et al.,1998). The ligand activation of PPARγ causes a remarkable response inthe breast cancer cells with neutral lipid accumulation and changes ingene expression. 3. PPARγ is expressed at high levels in humanliposarcoma, and primary liposarcoma cells can be induced to undergoterminal differentiation by treatment with the PPARγ ligandpioglitazone, demonstrating that the differentiation block in thesecells can be overcome by maximal activation of the PPARγ pathway(Tontonoz et al., 1997). Remarkably, simultaneous treatment ofliposarcoma cells with both PPARγ- and RXR-specific ligands resulted inadditive stimulation of differentiation. 4. 15-deoxyΔ^(21,14)PGJ2, aPPARγ ligand, induced caspase-dependent apoptosis in JEG3choriocarcinoma cells (Keelan et al., 1999). 5. PPARγ2 transcript isexpressed in leukemic cells from patients with AML, ALL, and CML, aswell as in normal neutrophils and peripheral blood lymphocytes (Greeneet al., 1995). In contrast, only full-length 1.85-kb PPARγl transcriptwas detected in a variety of human leukemia cell lines and in primarybone marrow stromal cells. The PPARγ gene is mapped to human chromosome3p25. 3p deletions are commonly seen in a variety of carcinomas, and the3p25-p21 deletion is seen infrequently in patients with chroniclymphocytic leukemias, and non-Hodgkin's lymphomas (Johansson et al.,1997). 6. PPARγ is also involved in angiogenesis (Veiga et al., 1999).Ligation of PPARγ exerted anti-angiogenic effects. Thus, theCDDO-compounds described herein, which are PPARγ ligands, provideselective killing of cancer cells that express more PPARγ receptors.Hence, the combination therapies described in this invention areenvisioned to be effective in various types of cancers.

[0100] D. Therapeutic Regimens

[0101] Tumor cell resistance to chemotherapy and radiotherapy agentsrepresents a major problem in clinical oncology. In the context of thepresent invention, it is contemplated that combination therapy usingCDDO-compounds with other chemotherapeutics could be used. The otherchemotherapeutics include retinoid compounds described above and inother parts of this specification and include RXR-specific ligands,ATRA, 9-cis-retinoic acid, LG100268, LGD1069 (Targretin, bexarotene),fenretinide [N-(4-hydroxyphenyl)retinamide, 4-HPR], and CD437. Inaddition other synthetic and naturally occurring retinoids arecontemplated as useful. As the CDDO-compounds are PPARγ ligands and theheterodimerization of PPARγ and RXR's enhances gene transcription ofapoptotic pathways, other PPARγ ligands such as those described aboveand in other parts of this specification are also contemplated as usefulanticancer therapies in combination with retinoids. Some examples areendogenous PPARγ ligands such as 15-deoxyΔ^(12,14)PGJ2 and linoleic acidand pharmaceutical PPARγ ligands including the thiazolidinediones (TZDs)such as troglitazone, BRL49653 (rosiglitazone), and pioglitazone,L-805645, GW347845X, and non-steroidal anti-inflammatory drugs. Inaddition, other PPARγ-ligands contemplated as useful in this inventioninclude all naturally occurring ligands as well as syntheticallyprepared compounds. Yet other chemotherapeutic agents that may be usedin combination with the CDDO-compounds to achieve cancer therapy aredescribed later in the specification.

[0102] Cancers that can be treated with the present invention include,but are not limited to, hematological malignancies including: bloodcancer, myeloid leukemia, monocytic leukemia, myelocytic leukemia,promyelocytic leukemia, myeloblastic leukemia, lymphocytic leukemia,acute myelogenous leukemic, chronic myelogenous leukemic, lymphoblasticleukemia, hairy cell leukemia. Solid cell tumors and cancers that can betreated include those such as tumors of the brain (glioblastomas,medulloblastoma, astrocytoma, oligodendroglioma, ependymomas), lung,liver, spleen, kidney, lymph node, small intestine, pancreas, colon,stomach, breast, endometrium, prostate, testicle, ovary, skin, head andneck, esophagus. Furthermore, the cancer may be a precancer, ametastatic and/or a non-metastatic cancer.

[0103] “Effective amount” is defined as an amount of the agent that willdecrease, reduce, inhibit or otherwise abrogate the growth of a cancercell, induce apoptosis, inhibit metastasis, induce differentation, killcells or induce cytotoxicity in cells.

[0104] The administration of the other chemotherapeutic may precede orfollow the therapy using CDDO-compounds by intervals ranging fromminutes to days to weeks. In embodiments where the otherchemotherapeutic and the CDDO-compound are administered together, onewould generally ensure that a significant period of time did not expirebetween the time of each delivery. In such instances, it is contemplatedthat one would administer to a patient both modalities within about12-24 hours of each other and, more preferably, within about 6-12 hoursof each other, with a delay time of only about 12 hours being mostpreferred. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several days (2, 3,4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse betweenthe respective administrations.

[0105] It also is conceivable that more than one administration ofeither the other chemotherapeutic and the CDDO-compound will be requiredto achieve complete cancer cure. Various combinations may be employed,where the other chemotherapeutic agent is “A” and the CDDO-compound is“B”, as exemplified below: A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/AB/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/BB/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

[0106] Other combinations also are contemplated. The exact dosages andregimens of each agent can be suitable altered by those of ordinaryskill in the art.

[0107] In embodiments of the invention that concern graft versus hostdisease (GVHD) treatment and prevention, the additional use ofimmunosupressive agents is also contemplated. Thus, ‘A’ in the abovescheme may represent an immunosupressive agent. Furthermore, it is alsocontemplated that one may use a CDDO-compound, a chemotherapeutic agentas well as an immunosupressive agent in various combinations.

[0108] a) Chemotherapeutic Agents

[0109] Agents that damage DNA are chemotherapeutics. These can be, forexample, agents that directly cross-link DNA, agents that intercalateinto DNA, and agents that lead to chromosomal and mitotic aberrations byaffecting nucleic acid synthesis. Agents that directly cross-linknucleic acids, specifically DNA, are envisaged and are exemplified bycisplatin, and other DNA alkylating agents. Agents that damage DNA alsoinclude compounds that interfere with DNA replication, mitosis, andchromosomal segregation.

[0110] Some examples of chemotherapeutic agents include antibioticchemotherapeutics such as, Doxorubicin, Daunorubicin, Mitomycin (alsoknown as mutamycin and/or mitomycin-C), Actinomycin D (Dactinomycin),Bleomycin, Plicomycin, Plant alkaloids such as Taxol, Vincristine,Vinblastine. Miscellaneous agents such as Cisplatin, VP16, TumorNecrosis Factor. Alkylating Agents such as, Carmustine, Melphalan (alsoknown as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM,or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard),Cyclophosphamide, Chlorambucil, Busulfan (also known as myleran),Lomustine. And other agents for example, Cisplatin (CDDP), Carboplatin,Procarbazine, Mechlorethamine, Camptothecin, Ifosfamide, Nitrosurea,Etoposide (VP16), Tamoxifen, Raloxifene, Estrogen Receptor BindingAgents, Gemcitabien, Navelbine, Farnesyl-protein transferase inhibitors,Transplatinum, 5-Fluorouracil, and Methotrexate, Temazolomide (anaqueous form of DTIC), Mylotarg, Dolastatin-10, Bryostatin, or anyanalog or derivative variant of the foregoing.

[0111] Retinoids and the PPARγ ligands are also some chemotherapeuticagents contemplated useful in the present invention. Retinoids includeall RXR and RAR-specific retinoic acid ligands. For example, ATRA, 9-cisretinoic acid, LG100268, LGD1069 (Targretin, bexarotene), fenretinide[N-(4-hydroxyphenyl)retinamide, 4-HPR] and CD437 are contemplated asuseful. CD437 is a novel retinoids that binds to the RARγ and RARβretinoids receptors. It is a potent inducer of apoptosis in vitro. Notrials in humans have been conducted with this chemotherapeutic so far.However, in mice models, oral administration of 10-30 mg/kg daily for 3wk or injection of 10 mg/kg of body weight in the tumor caused growthinhibition of melanoma xenografts in vivo (Schadendorf D. et al., 1996).

[0112] ATRA may be administered at a range of 10-100 mg/m²/day, forexample, at 45 mg/m²/day orally daily. A liposomal formulation of ATRAmay be administered at 90 mg/m²/day IV. 9-cis-Retinoid acid may beadministered at a range of 20-150 mg/m² twice a day orally. LG100268 inmice models was administered at a dose of 5-10 mg/kg. LGD1069 iscontemplated as useful for the topical treatment of cutaneous lesions inpatients with cutaneous T-cell lymphoma (CTCL) who have refractory orresistant disease after other therapies. In some embodiments the LGD1069is administered as capsules of 300-400 mg/m²/day taken orally. In otherembodiments, LGD1069 is administered as a gel of about 1% strength forthe topical treatment of cutaneous lesions in patients with CTCL (Stage(1A and 1B) who have refractory or resistant disease after other cancertherapies and may be taken two to four times daily. Fenretinide[N-(4-hydroxyphenyl)retinamide, 4-HPR] is contemplated useful at 25-600mg daily and may be administered continuously in some embodiments.

[0113] Endogenous PPARγ ligands such as 15-deoxyΔ^(12,14)PGJ2 andlinoleic acid and pharmaceutical PPARγ ligands including thethiazolidinediones (TZDs) such as troglitazone, BRL49653 (rosiglitazone)and pioglitazone, L-805645, GW347845X, and non-steroidalanti-inflammatory drugs are also contemplated useful in context of theinstant invention.

[0114] (i) Antibiotics

[0115] Doxorubicin. Doxorubicin hydrochloride, 5,12-Naphthacenedione,(8s-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-hydrochloride(hydroxydaunorubicin hydrochloride, Adriamycin) is used in a wideantineoplastic spectrum. It binds to DNA and inhibits nucleic acidsynthesis, inhibits mitosis and promotes chromosomal aberrations.

[0116] Administered alone, it is the drug of first choice for thetreatment of thyroid adenoma and primary hepatocellular carcinoma. It isa component of 31 first-choice combinations for the treatment ofovarian, endometrial and breast tumors, bronchogenic oat-cell carcinoma,non-small cell lung carcinoma, gastric adenocarcinoma, retinoblastoma,neuroblastoma, mycosis fungoides, pancreatic carcinoma, prostaticcarcinoma, bladder carcinoma, myeloma, diffuse histiocytic lymphoma,Wilms' tumor, Hodgkin's disease, adrenal tumors, osteogenic sarcoma softtissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma and acute lymphocyticleukemia. It is an alternative drug for the treatment of islet cell,cervical, testicular and adrenocortical cancers. It is also animmunosuppressant.

[0117] Doxorubicin is absorbed poorly and must be administeredintravenously. The pharmacokinetics are multicompartmental. Distributionphases have half-lives of 12 minutes and 3.3 hr. The eliminationhalf-life is about 30 hr. Forty to 50% is secreted into the bile. Mostof the remainder is metabolized in the liver, partly to an activemetabolite (doxorubicinol), but a few percent is excreted into theurine. In the presence of liver impairment, the dose should be reduced.

[0118] Appropriate doses are, intravenous, adult, 60 to 75 mg/m² at21-day intervals or 25 to 30 mg/m² on each of 2 or 3 successive daysrepeated at 3- or 4-wk intervals or 20 mg/m² once a week. The lowestdose should be used in elderly patients, when there is prior bone-marrowdepression caused by prior chemotherapy or neoplastic marrow invasion,or when the drug is combined with other myelopoietic suppressant drugs.The dose should be reduced by 50% if the serum bilirubin lies between1.2 and 3 mg/dL and by 75% if above 3 mg/dL. The lifetime total doseshould not exceed 550 mg/m² in patients with normal heart function and400 mg/m² in persons having received mediastinal irradiation.Alternatively, 30 mg/m² on each of 3 consecutive days, repeated every 4wk. Exemplary doses may be 10 mg/m ², 20 mg/m², 30 mg/m², 50 mg/m², 100mg/m², 150 mg/m², 175 mg/m², 200 mg/m², 225 mg/m², 250 mg/m², 275 mg/m²,300 mg/m², 350 mg/m², 400 mg/m², 425 mg/m², 450 mg/m², 475 mg/m², 500mg/m². Of course, all of these dosages are exemplary, and any dosagein-between these points is also expected to be of use in the invention.

[0119] Daunorubicin. Daunorubicin hydrochloride, 5,12-Naphthacenedione,(8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexanopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-,hydrochloride; also termed cerubidine and available from Wyeth.Daunorubicin intercalates into DNA, blocks DAN-directed RNA polymeraseand inhibits DNA synthesis. It can prevent cell division in doses thatdo not interfere with nucleic acid synthesis.

[0120] In combination with other drugs it is included in thefirst-choice chemotherapy of acute myelocytic leukemia in adults (forinduction of remission), acute lymphocytic leukemia and the acute phaseof chronic myelocytic leukemia. Oral absorption is poor, and it must begiven intravenously. The half-life of distribution is 45 minutes and ofelimination, about 19 hr. The half-life of its active metabolite,daunorubicinol, is about 27 hr. Daunorubicin is metabolized mostly inthe liver and also secreted into the bile (ca 40%). Dosage must bereduced in liver or renal insufficiencies.

[0121] Suitable doses are (base equivalent), intravenous adult, youngerthan 60 yr. 45 mg/m²/day (30 mg/m² for patients older than 60 yr.) for1, 2 or 3 days every 3 or 4 wk or 0.8 mg/kg/day for 3 to 6 days every 3or 4 wk; no more than 550 mg/m² should be given in a lifetime, exceptonly 450 mg/m² if there has been chest irradiation; children, 25 mg/m²once a week unless the age is less than 2 yr. or the body surface lessthan 0.5 m, in which case the weight-based adult schedule is used. It isavailable in injectable dosage forms (base equivalent) 20 mg (as thebase equivalent to 21.4 mg of the hydrochloride). Exemplary doses may be10 mg/m², 20 mg/m², 30 mg/m², 50 mg/m², 100 mg/m², 150 mg/m², 175 mg/m²,200 mg/m², 225 mg/m², 250 mg/m², 275 mg/m², 300 mg/m², 350 mg/m², 400mg/m², 425 mg/m², 450 mg/m², 475 mg/m², 500 mg/m². Of course, all ofthese dosages are exemplary, and any dosage in-between these points isalso expected to be of use in the invention.

[0122] Mitomycin. Mitomycin (also known as mutamycin and/or mitomycin-C)is an antibiotic isolated from the broth of Streptomyces caespitosuswhich has been shown to have antitumor activity. The compound is heatstable, has a high melting point, and is freely soluble in organicsolvents.

[0123] Mitomycin selectively inhibits the synthesis of deoxyribonucleicacid (DNA). The guanine and cytosine content correlates with the degreeof mitomycin-induced cross-linking. At high concentrations of the drug,cellular RNA and protein synthesis are also suppressed.

[0124] In humans, mitomycin is rapidly cleared from the serum afterintravenous administration. Time required to reduce the serumconcentration by 50% after a 30 mg bolus injection is 17 minutes. Afterinjection of 30 mg, 20 mg, or 10 mg I.V., the maximal serumconcentrations were 2.4 mg/mL, 1.7 mg/mL, and 0.52 mg/mL, respectively.Clearance is effected primarily by metabolism in the liver, butmetabolism occurs in other tissues as well. The rate of clearance isinversely proportional to the maximal serum concentration because, it isthought, of saturation of the degradative pathways.

[0125] Approximately 10% of a dose of mitomycin is excreted unchanged inthe urine. Since metabolic pathways are saturated at relatively lowdoses, the percent of a dose excreted in urine increases with increasingdose. In children, excretion of intravenously administered mitomycin issimilar.

[0126] Actinomycin D. Actinomycin D (Dactinomycin) [50-76-0];C₆₂H₈₆N₁₂O₁₆ (1255.43) is an antineoplastic drug that inhibitsDNA-dependent RNA polymerase. It is a component of first-choicecombinations for treatment of choriocarcinoma, embryonalrhabdomyosarcoma, testicular tumor and Wilms' tumor. Tumors which failto respond to systemic treatment sometimes respond to local perfusion.Dactinomycin potentiates radiotherapy. It is a secondary (efferent)immunosuppressive.

[0127] Actinomycin D is used in combination with primary surgery,radiotherapy, and other drugs, particularly vincristine andcyclophosphamide. Antineoplastic activity has also been noted in Ewing'stumor, Kaposi's sarcoma, and soft-tissue sarcomas. Dactinomycin can beeffective in women with advanced cases of choriocarcinoma. It alsoproduces consistent responses in combination with chlorambucil andmethotrexate in patients with metastatic testicular carcinomas. Aresponse may sometimes be observed in patients with Hodgkin's diseaseand non-Hodgkin's lymphomas. Dactinomycin has also been used to inhibitimmunological responses, particularly the rejection of renaltransplants.

[0128] Half of the dose is excreted intact into the bile and 10% intothe urine; the half-life is about 36 hr. The drug does not pass theblood-brain barrier. Actinomycin D is supplied as a lyophilized powder(0/5 mg in each vial). The usual daily dose is 10 to 15 mg/kg; this isgiven intravenously for 5 days; if no manifestations of toxicity areencountered, additional courses may be given at intervals of 3 to 4weeks. Daily injections of 100 to 400 mg have been given to children for10 to 14 days; in other regimens, 3 to 6 mg/kg, for a total of 125mg/kg, and weekly maintenance doses of 7.5 mg/kg have been used.Although it is safer to administer the drug into the tubing of anintravenous infusion, direct intravenous injections have been given,with the precaution of discarding the needle used to withdraw the drugfrom the vial in order to avoid subcutaneous reaction. Exemplary dosesmay be 100 mg/m, 150 mg/m², 175 mg/m², 200 mg/m², 225 mg/m², 250 mg/m²,275 mg/m², 300 mg/m², 350 mg/m², 400 mg/m², 425 mg/m², 450 mg/m², 475mg/m², 500 mg/m². Of course, all of these dosages are exemplary, and anydosage in-between these points is also expected to be of use in theinvention.

[0129] Bleomycin. Bleomycin is a mixture of cytotoxic glycopeptideantibiotics isolated from a strain of Streptomyces verticillus. It isfreely soluble in water. Although the exact mechanism of action ofbleomycin is unknown, available evidence would seem to indicate that themain mode of action is the inhibition of DNA synthesis with someevidence of lesser inhibition of RNA and protein synthesis.

[0130] In mice, high concentrations of bleomycin are found in the skin,lungs, kidneys, peritoneum, and lymphatics. Tumor cells of the skin andlungs have been found to have high concentrations of bleomycin incontrast to the low concentrations found in hematopoietic tissue. Thelow concentrations of bleomycin found in bone marrow may be related tohigh levels of bleomycin degradative enzymes found in that tissue.

[0131] In patients with a creatinine clearance of >35 mL per minute, theserum or plasma terminal elimination half-life of bleomycin isapproximately 115 minutes. In patients with a creatinine clearance of<35 mL per minute, the plasma or serum terminal elimination half-lifeincreases exponentially as the creatinine clearance decreases. Inhumans, 60% to 70% of an administered dose is recovered in the urine asactive bleomycin.

[0132] Bleomycin should be considered a palliative treatment. It hasbeen shown to be useful in the management of the following neoplasmseither as a single agent or in proven combinations with other approvedchemotherapeutic agents in squamous cell carcinoma such as head and neck(including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus,palate, lip, buccal mucosa, gingiva, epiglottis, larynx), skin, penis,cervix, and vulva. It has also been used in the treatment of lymphomasand testicular carcinoma.

[0133] Because of the possibility of an anaphylactoid reaction, lymphomapatients should be treated with two units or less for the first twodoses. If no acute reaction occurs, then the regular dosage schedule maybe followed.

[0134] Improvement of Hodgkin's Disease and testicular tumors is promptand noted within 2 weeks. If no improvement is seen by this time,improvement is unlikely. Squamous cell cancers respond more slowly,sometimes requiring as long as 3 weeks before any improvement is noted.

[0135] Bleomycin may be given by the intramuscular, intravenous, orsubcutaneous routes.

[0136] (ii) Miscellaneous Agents

[0137] Cisplatin. Cisplatin has been widely used to treat cancers suchas metastatic testicular or ovarian carcinoma, advanced bladder cancer,head or neck cancer, cervical cancer, lung cancer or other tumors.Cisplatin can be used alone or in combination with other agents, withefficacious doses used in clinical applications of 15-20 mg/m² for 5days every three weeks for a total of three courses. Exemplary doses maybe 0.50 mg/m², 1.0 mg/m², 1.50 mg/m², 1.75 mg/m², 2.0 mg/m², 3.0 mg/m²,4.0 mg/m², 5.0 mg/m², 10 mg//m². Of course, all of these dosages areexemplary, and any dosage in-between these points is also expected to beof use in the invention.

[0138] Cisplatin is not absorbed orally and must therefore be deliveredvia injection intravenously, subcutaneously, intratumorally orintraperitoneally.

[0139] Cisplatin may also be used in combination with emodin oremodin-like compounds in the treatment of non-small cell lung carcinoma.Combination of cisplatin and emodin and or emodin-like compounds mayalso be used for the treatment of any neu-mediated cancers.

[0140] VP16. VP16 is also know as etoposide and is used primarily fortreatment of testicular tumors, in combination with bleomycin andcisplatin, and in combination with cisplatin for small-cell carcinoma ofthe lung. It is also active against non-Hodgkin's lymphomas, acutenonlymphocytic leukemia, carcinoma of the breast, and Kaposi's sarcomaassociated with acquired immunodeficiency syndrome (AIDS).

[0141] VP16 is available as a solution (20 mg/ml) for intravenousadministration and as 50-mg, liquid-filled capsules for oral use. Forsmall-cell carcinoma of the lung, the intravenous dose (in combinationtherapy) is can be as much as 100 mg/m ² or as little as 2 mg/ m²,routinely 35 mg/m², daily for 4 days, to 50 mg/m², daily for 5 days havealso been used. When given orally, the dose should be doubled. Hence thedoses for small cell lung carcinoma may be as high as 200-250 mg/m². Theintravenous dose for testicular cancer (in combination therapy) is 50 to100 mg/m² daily for 5 days, or 100 mg/m² on alternate days, for threedoses. Cycles of therapy are usually repeated every 3 to 4 weeks. Thedrug should be administered slowly during a 30- to 60-minute infusion inorder to avoid hypotension and bronchospasm, which are probably due tothe solvents used in the formulation.

[0142] Tumor Necrosis Factor. Tumor Necrosis Factor [TNF; Cachectin] isa glycoprotein that kills some kinds of cancer cells, activates cytokineproduction, activates macrophages and endothelial cells, promotes theproduction of collagen and collagenases, is an inflammatory mediator andalso a mediator of septic shock, and promotes catabolism, fever andsleep. Some infectious agents cause tumor regression through thestimulation of TNF production. TNF can be quite toxic when used alone ineffective doses, so that the optimal regimens probably will use it inlower doses in combination with other drugs. Its immunosuppressiveactions are potentiated by gamma-interferon, so that the combinationpotentially is dangerous. A hybrid of TNF and interferon-α also has beenfound to possess anti-cancer activity.

[0143] (iii) Plant Alkaloids

[0144] Taxol. Taxol is an experimental antimitotic agent, isolated fromthe bark of the ash tree, Taxus brevifolia. It binds to tubulin (at asite distinct from that used by the vinca alkaloids) and promotes theassembly of microtubules. Taxol is currently being evaluated clinically;it has activity against malignant melanoma and carcinoma of the ovary.Maximal doses are 30 mg/m² per day for 5 days or 210 to 250 mg/m² givenonce every 3 weeks. Of course, all of these dosages are exemplary, andany dosage in-between these points is also expected to be of use in theinvention.

[0145] Vincristine. Vincristine blocks mitosis and produces metaphasearrest. It seems likely that most of the biological activities of thisdrug can be explained by its ability to bind specifically to tubulin andto block the ability of protein to polymerize into microtubules. Throughdisruption of the microtubules of the mitotic apparatus, cell divisionis arrested in metaphase. The inability to segregate chromosomescorrectly during mitosis presumably leads to cell death.

[0146] The relatively low toxicity of vincristine for normal marrowcells and epithelial cells make this agent unusual among anti-neoplasticdrugs, and it is often included in combination with othermyelosuppressive agents.

[0147] Unpredictable absorption has been reported after oraladministration of vinblastine or vincristine. At the usual clinicaldoses the peak concentration of each drug in plasma is approximately 0.4mM.

[0148] Vinblastine and vincristine bind to plasma proteins. They areextensively concentrated in platelets and to a lesser extent inleukocytes and erythrocytes.

[0149] Vincristine has a multiphasic pattern of clearance from theplasma; the terminal half-life is about 24 hours. The drug ismetabolized in the liver, but no biologically active derivatives havebeen identified. Doses should be reduced in patients with hepaticdysfunction. At least a 50% reduction in dosage is indicated if theconcentration of bilirubin in plasma is greater than 3 mg/dl (about 50mM).

[0150] Vincristine sulfate is available as a solution (1 mg/ml) forintravenous injection. Vincristine used together with corticosteroids ispresently the treatment of choice to induce remissions in childhoodleukemia; the optimal dosages for these drugs appear to be vincristine,intravenously, 2 mg/m² of body-surface area, weekly, and prednisolone,orally, 40 mg/m², daily. Adult patients with Hodgkin's disease ornon-Hodgkin's lymphomas usually receive vincristine as a part of acomplex protocol. When used in the MOPP regimen, the recommended dose ofvincristine is 1.4 mg/m². High doses of vincristine seem to be toleratedbetter by children with leukemia than by adults, who may experiencesever neurological toxicity. Administration of the drug more frequentlythan every 7 days or at higher doses seems to increase the toxicmanifestations without proportional improvement in the response rate.Precautions should also be used to avoid extravasation duringintravenous administration of vincristine. Vincristine (and vinblastine)can be infused into the arterial blood supply of tumors in doses severaltimes larger than those that can be administered intravenously withcomparable toxicity.

[0151] Vincristine has been effective in Hodgkin's disease and otherlymphomas. Although it appears to be somewhat less beneficial thanvinblastine when used alone in Hodgkin's disease, when used withmechlorethamine, prednisolone, and procarbazine (the so-called MOPPregimen), it is the preferred treatment for the advanced stages (III andIV) of this disease. In non-Hodgkin's lymphomas, vincristine is animportant agent, particularly when used with cyclophosphamide,bleomycin, doxorubicin, and prednisolone. Vincristine is more usefulthan vinblastine in lymphocytic leukemia. Beneficial response have beenreported in patients with a variety of other neoplasms, particularlyWilms' tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, andcarcinomas of the breast, bladder, and the male and female reproductivesystems.

[0152] Doses of vincristine for use will be determined by the clinicianaccording to the individual patients need. 0.01 to 0.03 mg/kg or 0.4 to1.4 mg/m² can be administered or 1.5 to 2 mg/m² can also beadministered. Alternatively 0.02 mg/m², 0.05 mg/m², 0.06 mg/m², 0.07mg/m, 0.08 mg/m², 0.1 mg/m², 0.12 mg/m, 0.14 mg/m², 0.15 mg/m², 0.2mg/m², 0.25 mg/m² can be given as a constant intravenous infusion. Ofcourse, all of these dosages are exemplary, and any dosage in-betweenthese points is also expected to be of use in the invention.

[0153] Vinblastine. When cells are incubated with vinblastine,dissolution of the microtubules occurs. Unpredictable absorption hasbeen reported after oral administration of vinblastine or vincristine.At the usual clinical doses the peak concentration of each drug inplasma is approximately 0.4 mM. Vinblastine and vincristine bind toplasma proteins. They are extensively concentrated in platelets and to alesser extent in leukocytes and erythrocytes.

[0154] After intravenous injection, vinblastine has a multiphasicpattern of clearance from the plasma; after distribution, drugdisappears from plasma with half-lives of approximately 1 and 20 hours.

[0155] Vinblastine is metabolized in the liver to biologically activatederivative desacetylvinblastine. Approximately 15% of an administereddose is detected intact in the urine, and about 10% is recovered in thefeces after biliary excretion. Doses should be reduced in patients withhepatic dysfunction. At least a 50% reduction in dosage is indicated ifthe concentration of bilirubin in plasma is greater than 3 mg/dl (about50 mM).

[0156] Vinblastine sulfate is available in preparations for injection.The drug is given intravenously; special precautions must be takenagainst subcutaneous extravasation, since this may cause painfulirritation and ulceration. The drug should not be injected into anextremity with impaired circulation. After a single dose of 0.3 mg/kg ofbody weight, myelosuppression reaches its maximum in 7 to 10 days. If amoderate level of leukopenia (approximately 3000 cells/mm³) is notattained, the weekly dose may be increased gradually by increments of0.05 mg/kg of body weight. In regimens designed to cure testicularcancer, vinblastine is used in doses of 0.3 mg/kg every 3 weeksirrespective of blood cell counts or toxicity.

[0157] The most important clinical use of vinblastine is with bleomycinand cisplatin in the curative therapy of metastatic testicular tumors.Beneficial responses have been reported in various lymphomas,particularly Hodgkin's disease, where significant improvement may benoted in 50 to 90% of cases. The effectiveness of vinblastine in a highproportion of lymphomas is not diminished when the disease is refractoryto alkylating agents. It is also active in Kaposi's sarcoma,neuroblastoma, and Letterer-Siwe disease (histiocytosis X), as well asin carcinoma of the breast and choriocarcinoma in women.

[0158] Doses of vinblastine for use will be determined by the clinicianaccording to the individual patients need. 0.1 to 0.3 mg/kg can beadministered or 1.5 to 2 mg/m² can also be administered. Alternatively,0.1 mg/m², 0.12 mg/m², 0.14 mg/m², 0.15 mg/m², 0.2 mg/m², 0.25 mg/m²,0.5 mg/m², 1.0 mg/m², 1.2 mg/m², 1.4 mg/m², 1.5 mg/m², 2.0 mg/m², 2.5mg/m², 5.0 mg/m², 6 mg/m², 8 mg/m², 9 mg/m², 10 mg/m², 20 mg/m², can begiven. Of course, all of these dosages are exemplary, and any dosagein-between these points is also expected to be of use in the invention.

[0159] (iv) Alkylating Agents

[0160] Carmustine. Carmustine (sterile carmustine) is one of thenitrosoureas used in the treatment of certain neoplastic diseases. It is1,3bis (2-chloroethyl)-1-nitrosourea. It is lyophilized pale yellowflakes or congealed mass with a molecular weight of 214.06. It is highlysoluble in alcohol and lipids, and poorly soluble in water. Carmustineis administered by intravenous infusion after reconstitution asrecommended. Sterile carmustine is commonly available in 100 mg singledose vials of lyophilized material.

[0161] Although it is generally agreed that carmustine alkylates DNA andRNA, it is not cross resistant with other alkylators. As with othernitrosoureas, it may also inhibit several key enzymatic processes bycarbamoylation of amino acids in proteins.

[0162] Carmustine is indicated as palliative therapy as a single agentor in established combination therapy with other approvedchemotherapeutic agents in brain tumors such as glioblastoma, brainstemglioma, medullobladyoma, astrocytoma, ependymoma, and metastatic braintumors. Also it has been used in combination with prednisolone to treatmultiple myeloma. Carmustine has proved useful, in the treatment ofHodgkin's Disease and in non-Hodgkin's lymphomas, as secondary therapyin combination with other approved drugs in patients who relapse whilebeing treated with primary therapy, or who fail to respond to primarytherapy.

[0163] The recommended dose of carmustine as a single agent inpreviously untreated patients is 150 to 200 mg/m² intravenously every 6weeks. This may be given as a single dose or divided into dailyinjections such as 75 to 100 mg/m² on 2 successive days. When carmustineis used in combination with other myelosuppressive drugs or in patientsin whom bone marrow reserve is depleted, the doses should be adjustedaccordingly. Doses subsequent to the initial dose should be adjustedaccording to the hematologic response of the patient to the precedingdose. It is of course understood that other doses may be used in thepresent invention for example 10 mg/m², 20 mg/m², 30 mg/m² 40 mg/m² 50mg/m² 60 mg/m² 70 mg/m² 80 mg/m² 90 mg/m²100 mg/m². The skilled artisanis directed to, “Remington's Pharmaceutical Sciences” 15th Edition,chapter 61. Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject

[0164] Melphalan. Melphalan also known as alkeran, L-phenylalaninemustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is aphenylalanine derivative of nitrogen mustard. Melphalan is abifunctional alkylating agent which is active against selective humanneoplastic diseases. It is known chemically as4-[bis(2-chloroethyl)amino]-L-phenylalanine.

[0165] Melphalan is the active L-isomer of the compound and was firstsynthesized in 1953 by Bergel and Stock; the D-isomer, known asmedphalan, is less active against certain animal tumors, and the doseneeded to produce effects on chromosomes is larger than that requiredwith the L-isomer. The racemic (DL-) form is known as merphalan orsarcolysin. Melphalan is insoluble in water and has a pKa₁ of ˜2.1.Melphalan is available in tablet form for oral administration and hasbeen used to treat multiple myeloma.

[0166] Available evidence suggests that about one third to one half ofthe patients with multiple myeloma show a favorable response to oraladministration of the drug.

[0167] Melphalan has been used in the treatment of epithelial ovariancarcinoma. One commonly employed regimen for the treatment of ovariancarcinoma has been to administer melphalan at a dose of 0.2 mg/kg dailyfor five days as a single course. Courses are repeated every four tofive weeks depending upon hematologic tolerance (Smith and Rutledge,1975; Young et al., 1978). Alternatively the dose of melphalan usedcould be as low as 0.05 mg/kg/day or as high as 3 mg/kg/day or any dosein between these doses or above these doses. Some variation in dosagewill necessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject.

[0168] Cyclophosphamide. Cyclophosphamide is2H-1,3,2-Oxazaphosphorin-2-amine, N,N-bis(2-chloroethyl)tetrahydro-,2-oxide, monohydrate; termed Cytoxan available from Mead Johnson; andNeosar available from Adria. Cyclophosphamide is prepared by condensing3-amino-1-propanol with N,N-bis(2-chlorethyl) phosphoramidic dichloride[(ClCH₂CH₂)₂N-POCl₂] in dioxane solution under the catalytic influenceof triethylamine. The condensation is double, involving both thehydroxyl and the amino groups, thus effecting the cyclization.

[0169] Unlike other β-chloroethylamino alkylators, it does not cyclizereadily to the active ethyleneimonium form until activated by hepaticenzymes. Thus, the substance is stable in the gastrointestinal tract,tolerated well and effective by the oral and parental routes and doesnot cause local vesication, necrosis, phlebitis or even pain.

[0170] Suitable doses for adults include, orally, 1 to 5 mg/kg/day(usually in combination), depending upon gastrointestinal tolerance; or1 to 2 mg/kg/day; intravenously, initially 40 to 50 mg/kg in divideddoses over a period of 2 to 5 days or 10 to 15 mg/kg every 7 to 10 daysor 3 to 5 mg/kg twice a week or 1.5 to 3 mg/kg/day. A dose 250 mg/kg/daymay be administered as an antineoplastic. Because of gastrointestinaladverse effects, the intravenous route is preferred for loading. Duringmaintenance, a leukocyte count of 3000 to 4000/mm³ usually is desired.The drug also sometimes is administered intramuscularly, by infiltrationor into body cavities. It is available in dosage forms for injection of100, 200 and 500 mg, and tablets of 25 and 50 mg the skilled artisan isreferred to “Remington's Pharmaceutical Sciences” 15th Edition, chapter61, incorporate herein as a reference, for details on doses foradministration.

[0171] Chlorambucil. Chlorambucil (also known as leukeran) was firstsynthesized by Everett et al. (1953). It is a bifunctional alkylatingagent of the nitrogen mustard type that has been found active againstselected human neoplastic diseases. Chlorambucil is known chemically as4-[bis(2-chlorethyl)amino] benzenebutanoic acid.

[0172] Chlorambucil is available in tablet form for oral administration.It is rapidly and completely absorbed from the gastrointestinal tract.After single oral doses of 0.6-1.2 mg/kg, peak plasma chlorambucillevels are reached within one hour and the terminal half-life of theparent drug is estimated at 1.5 hours. 0.1 to 0.2 mg/kg/day or 3 to 6mg/m²/day or alternatively 0.4 mg/kg may be used for antineoplastictreatment. Treatment regimes are well know to those of skill in the artand can be found in the “Physicians Desk Reference” and in “RemingtonsPharmaceutical Sciences” referenced herein.

[0173] Chlorambucil is indicated in the treatment of chronic lymphatic(lymphocytic) leukemia, malignant lymphomas including lymphosarcoma,giant follicular lymphoma and Hodgkin's disease. It is not curative inany of these disorders but may produce clinically useful palliation.

[0174] Busulfan. Busulfan (also known as myleran) is a bifunctionalalkylating agent. Busulfan is known chemically as 1,4-butanedioldimethanesulfonate.

[0175] Busulfan is not a structural analog of the nitrogen mustards.Busulfan is available in tablet form for oral administration. Eachscored tablet contains 2 mg busulfan and the inactive ingredientsmagnesium stearate and sodium chloride.

[0176] Busulfan is indicated for the palliative treatment of chronicmyelogenous (myeloid, myelocytic, granulocytic) leukemia. Although notcurative, busulfan reduces the total granulocyte mass, relieves symptomsof the disease, and improves the clinical state of the patient.Approximately 90% of adults with previously untreated chronicmyelogenous leukemia will obtain hematologic remission with regressionor stabilization of organomegaly following the use of busulfan. It hasbeen shown to be superior to splenic irradiation with respect tosurvival times and maintenance of hemoglobin levels, and to beequivalent to irradiation at controlling splenomegaly.

[0177] Lomustine. Lomustine is one of the nitrosoureas used in thetreatment of certain neoplastic diseases. It is1-(2-chloro-ethyl)-3-cyclohexyl-1 nitrosourea. It is a yellow powderwith the empirical formula of C₉H₁₆C1N₃O₂ and a molecular weight of233.71. Lomustine is soluble in 10% ethanol (0.05 mg per mL) and inabsolute alcohol (70 mg per mL). Lomustine is relatively insoluble inwater (<0.05 mg per mL). It is relatively unionized at a physiologicalpH. Inactive ingredients in lomustine capsules are: magnesium stearateand mannitol.

[0178] Although it is generally agreed that lomustine alkylates DNA andRNA, it is not cross resistant with other alkylators. As with othernitrosoureas, it may also inhibit several key enzymatic processes bycarbamoylation of amino acids in proteins.

[0179] Lomustine may be given orally. Following oral administration ofradioactive lomustine at doses ranging from 30 mg/m² to 100 mg/m², abouthalf of the radioactivity given was excreted in the form of degradationproducts within 24 hours.

[0180] The serum half-life of the metabolites ranges from 16 hours to 2days. Tissue levels are comparable to plasma levels at 15 minutes afterintravenous administration.

[0181] Lomustine has been shown to be useful as a single agent inaddition to other treatment modalities, or in established combinationtherapy with other approved chemotherapeutic agents in both primary andmetastatic brain tumors, in patients who have already receivedappropriate surgical and/or radiotherapeutic procedures. It has alsoproved effective in secondary therapy against Hodgkin's Disease incombination with other approved drugs in patients who relapse whilebeing treated with primary therapy, or who fail to respond to primarytherapy.

[0182] The recommended dose of lomustine in adults and children as asingle agent in previously untreated patients is 130 mg/m² as a singleoral dose every 6 weeks. In individuals with compromised bone marrowfunction, the dose should be reduced to 100 mg/m² every 6 weeks. Whenlomustine is used in combination with other myelosuppressive drugs, thedoses should be adjusted accordingly. It is understood that other dosesmay be used for example, 20 mg/m², 30 mg/m², 40 mg/m², 50 mg/m², 60mg/m², 70 mg/m², 80 mg/m², 90 mg/m², 100 mg/m², 120 mg/m² or any dosesbetween these figures as determined by the clinician to be necessary forthe individual being treated.

[0183] E. Adjunct Cancer Therapies

[0184] In order to increase the effectiveness of the combination therapywith the CDDO-compounds as described in the present invention, it may bedesirable to combine these compositions with yet other agents effectivein the treatment of cancer such as but not limited to those describedbelow.

[0185] a) Radiotherapeutic Agents

[0186] Radiotherapeutic agents and factors include radiation and wavesthat induce DNA damage for example, γ-irradiation, X-rays,UV-irradiation, microwaves, electronic emissions, radioisotopes, and thelike. Therapy may be achieved by irradiating the localized tumor sitewith the above described forms of radiations. It is most likely that allof these factors effect a broad range of damage DNA, on the precursorsof DNA, the replication and repair of DNA, and the assembly andmaintenance of chromosomes.

[0187] Dosage ranges for X-rays range from daily doses of 50 to 200roentgens for prolonged periods of time (3 to 4 weeks), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells.

[0188] b) Surgery

[0189] Approximately 60% of persons with cancer will undergo surgery ofsome type, which includes preventative, diagnostic or staging, curativeand palliative surgery. Curative surgery is a cancer treatment that maybe used in conjunction with other therapies, such as the treatment ofthe present invention, chemotherapy, radiotherapy, hormonal therapy,gene therapy, immunotherapy and/or alternative therapies.

[0190] Curative surgery includes resection in which all or part ofcancerous tissue is physically removed, excised, and/or destroyed. Tumorresection refers to physical removal of at least part of a tumor. Inaddition to tumor resection, treatment by surgery includes lasersurgery, cryosurgery, electrosurgery, and miscopically controlledsurgery (Mohs' surgery). It is further contemplated that the presentinvention may be used in conjunction with removal of superficialcancers, precancers, or incidental amounts of normal tissue.

[0191] Upon excision of part of all of cancerous cells, tissue, ortumor, a cavity may be formed in the body. Treatment may be accomplishedby perfusion, direct injection or local application of the area with anadditional anti-cancer therapy. Such treatment may be repeated, forexample, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Thesetreatments may be of varying dosages as well.

[0192] c) Immunotherapy

[0193] Immunotherapeutics, generally, rely on the use of immune effectorcells and molecules to target and destroy cancer cells. The immuneeffector may be, for example, an antibody specific for some marker onthe surface of a tumor cell. The antibody alone may serve as an effectorof therapy or it may recruit other cells to actually effect cellkilling. The antibody also may be conjugated to a drug or toxin(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussistoxin, etc.) and serve merely as a targeting agent. Alternatively, theeffector may be a lymphocyte carrying a surface molecule that interacts,either directly or indirectly, with a tumor cell target. Variouseffector cells include cytotoxic T cells and NK cells. Immunotherapycould be used as part of a combined therapy, in conjunction with theCDDO-compounds-based therapy.

[0194] The general approach for combined therapy is discussed below. Inone aspect the immunotherapy can be used to target a tumor cell. Manytumor markers exist and any of these may be suitable for targeting inthe context of the present invention. Common tumor markers includecarcinoembryonic antigen, prostate specific antigen, urinary tumorassociated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG,Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155. Alternate immune stimulating molecules alsoexist including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN,chemokines such as MIP-1, MCP-1, IL-8 and growth factors such as FLT3ligand. Combining immune stimulating molecules, either as proteins orusing gene delivery in combination with the CDDO-compound basedcombination therapy of this invention will enhance anti-tumor effects.

[0195] (i) Passive Immunotherapy

[0196] A number of different approaches for passive immunotherapy ofcancer exist. They may be broadly categorized into the following:injection of antibodies alone; injection of antibodies coupled to toxinsor chemotherapeutic agents; injection of antibodies coupled toradioactive isotopes; injection of anti-idiotype antibodies; andfinally, purging of tumor cells in bone marrow.

[0197] (ii) Active Immunotherapy

[0198] In active immunotherapy, an antigenic peptide, polypeptide orprotein, or an autologous or allogenic tumor cell composition or“vaccine” is administered, generally with a distinct bacterial adjuvant(Ravindranath & Morton, 1991; Morton & Ravindranath, 1996; Morton etal., 1992; Mitchell et al., 1990; Mitchell et al., 1993).

[0199] (iii) Adoptive Immunotherapy

[0200] In adoptive immunotherapy, the patient's circulating lymphocytes,or tumor infiltrated lymphocytes, are isolated in vitro, activated bylymphokines such as IL-2 or transduced with genes for tumor necrosis,and readministered (Rosenberg et al., 1988; 1989). To achieve this, onewould administer to an animal, or human patient, an immunologicallyeffective amount of activated lymphocytes in combination with anadjuvant-incorporated antigenic peptide composition as described herein.The activated lymphocytes will most preferably be the patient's owncells that were earlier isolated from a blood or tumor sample andactivated (or “expanded”) in vitro.

[0201] d) Gene Therapy

[0202] In yet another embodiment, gene therapy in conjunction with thecombination therapy using the CDDO-compounds described in the inventionare contemplated. A variety of proteins are encompassed within theinvention, some of which are described below. Table 1 lists variousgenes that may be targeted for gene therapy of some form in combinationwith the present invention. TABLE 1 Gene Source Human Disease FunctionGrowth Factors FGF family member HST/KS Transfection INT-2 MMTV promoterFGF family member Insertion INTI/WNTI MMTV promoter Factor-likeInsertion SIS Simian sarcoma virus PDGF B Receptor Tyrosine KinasesERBB/HER Avian erythroblastosis Amplified, deleted EGF/TGF-α/ virus; ALVpromoter squamous cell Amphiregulin/ insertion; amplified cancer;glioblastoma Hetacellulin receptor human tumors ERBB-2/NEU/HER-2Transfected from rat Amplified breast, Regulated by NDF/ Glioblastomasovarian, gastric cancers Heregulin and EGF- Related factors FMS SMfeline sarcoma virus CSF-1 receptor KIT HZ feline sarcoma virusMGF/Steel receptor Hematopoicis TRK Transfection from NGF (nerve growthhuman colon cancer Factor) receptor MET Transfection from Scatterfactor/HGF human osteosarcoma Receptor RET Translocations and pointSporadic thyroid cancer; Orphan receptor Tyr mutations familialmedullary Kinase thyroid cancer; multiple endocrine neoplasias 2A and 2BROS URII avian sarcoma Orphan receptor Tyr Virus Kinase PDGF receptorTranslocation Chronic TEL(ETS-like Myelomonocytic transcription factor)/Leukemia PDGF receptor gene Fusion TGF-βreceptor Colon carcinomamismatch mutation target NONRECEPTOR TYROSINE KINASES ABI. Abelson Mul.V Chronic myelogenous Interact with RB, RNA leukemia translocationpolymerase, CRK, with BCR CBL FPS/FES Avian Fujinami SV;GA FeSV LCKMul.V (murine leukemia Src family; T cell virus) promoter signaling;interacts insertion CD4/CD8 T cells SRC Avian Rous sarcomaMembrane-associated Virus Tyr kinase with signaling function; activatedby receptor kinases YES Avian Y73 virus Src family; signaling SER/THRPROTEIN KINASES AKT AKT8 murine retrovirus Regulated by PI(3)K?;regulate 70-kd S6 k? MOS Maloney murine SV GVBD; cystostatic factor; MAPkinase kinase PIM-1 Promoter insertion Mouse RAF/MIL 3611 murine SV; MH2Signaling in RAS avian SV Pathway MISCELLANEOUS CELL SURFACE¹ APC Tumorsuppressor Colon cancer Interacts with catenins DCC Tumor suppressorColon cancer CAM domains E-cadherin Candidate tumor Breast cancerExtracellular homotypic Suppressor binding; intracellular interacts withcatenins PTC/NBCCS Tumor suppressor and Nevoid basal cell cancer 12transmembrane Drosophilia homology syndrome (Gorline domain; signalssyndrome) through Gli homogue CI to antagonize hedgehog pathway TAN-1Notch Translocation T-ALI. Signaling? homologue MISCELLANEOUS SIGNALINGBCL-2 Translocation B-cell lymphoma Apoptosis CBL Mu Cas NS-1 VTyrosine- Phosphorylated RING finger interact Abl CRK CT1010 ASV AdaptedSH2/SH3 interact Abl DPC4 Tumor suppressor Pancreatic cancerTGF-β-related signaling Pathway MAS Transfection and Possibleangiotensin Tumorigenicity Receptor NCK Adaptor SH2/SH3 GUANINENUCLEOTIDE EXCHANGERS AND BINDING PROTEINS BCR Translocated with ABLExchanger; protein in CML Kinase DBL Transfection Exchanger GSP NF-1Hereditary tumor Tumor suppressor RAS GAP Suppressor neurofibromatosisOST Transfection Exchanger Harvey-Kirsten, N-RAS HaRat SV; Ki RaSV;Point mutations in many Signal cascade Balb-MoMuSV; human tumorsTransfection VAV Transfection S112/S113; exchanger NUCLEAR PROTEINS ANDTRANSCRIPTION FACTORS BRCA1 Heritable suppressor Mammary Localizationunsettled cancer/ovarian cancer BRCA2 Heritable suppressor Mammarycancer Function unknown ERBA Avian erythroblastosis thyroid hormoneVirus receptor (transcription) ETS Avian E26 virus DNA binding EVII MuLVpromotor AML Transcription factor Insertion FOS FBI/FBR murine 1transcription factor osteosarcoma viruses with c-JUN GLI Amplifiedglioma Glioma Zinc finger; cubitus interruptus homologue is in hedgehogsignaling pathway; inhibitory link PTC and hedgehog HMGI/LIMTranslocation t(3:12) Lipoma Gene fusions high t(12:15) mobility groupHMGI-C (XT-hook) and transcription factor LIM or acidic domain JUNASV-17 Transcription factor AP-1 with FOS MLL/VHRK+ELI/MENTranslocation/fusion Acute myeloid leukemia Gene fusion of DNA- ELL withMLL binding and methyl Trithorax-like gene transferase MLL with ELI RNApol II elongation factor MYB Avian myeloblastosis DNA binding Virus MYCAvian MC29; Burkitt's lymphoma DNA binding with Translocation B-cell MAXpartner; cyclin Lymphomas; promoter regulation; interact Insertion avianRB?; regulate leukosis apoptosis? Virus N-MYC Amplified NeuroblastomaL-MYC Lung cancer REL Avian NF-κB family Retriculoendotheliosistranscription factor Virus SKI Avian SKV770 Transcription factorRetrovirus VHL Heritable suppressor Von Hippel-Landau Negative regulatoror syndrome elongin; transcriptional elongation complex WT-1 Wilm'stumor Transcription factor CELL CYCLE/DNA DAMAGE RESPONSE¹⁰⁻²¹ ATMHereditary disorder Ataxia-telangiectasia Protein/lipid kinase homology;DNA damage response upstream in P53 pathway BCL-2 TranslocationFollicular lymphoma Apoptosis FACC Point mutation Fanconi's anemia groupC (predisposition leukemia MDA-7 Fragile site 3p14.2 Lung carcinomaHistidine triad-related diadenosine 5′,3″″- tetraphosphate asymmetrichydrolase hMLI/MutL HNPCC Mismatch repair; MutL Homologue hMSH2/MutSHNPCC Mismatch repair; MutS Homologue hPMS1 HNPCC Mismatch repair; MutLHomologue hPMS2 HNPCC Mismatch repair; MutL Homologue INK4/MTS1 AdjacentINK-4B at Candidate MTS1 p16 CDK inhibitor 9p21; CDK complexessuppressor and MLM melanoma gene INK4B/MTS2 Candidate suppressor p15 CDKinhibitor MDM-2 Amplified Sarcoma Negative regulator p53 p53 Associationwith SV40 Mutated > 50% human Transcription factor; T antigen tumors,including checkpoint control; hereditary Li-Fraumeni apoptosis syndromePRAD1/BCL1 Translocation with Parathyroid adenoma; Cyclin D Parathyroidhormone B-CLL or IgG RB Hereditary Retinoblastoma; Interact cyclin/cdk;Retinoblastoma; osteosarcoma; breast regulate E2F Association with manycancer; other sporadic transcription factor DNA virus tumor cancersAntigens XPA xerodema Excision repair; photo- pigmentosum; skin productrecognition; cancer predisposition zinc finger

[0203] e) Other Agents

[0204] It is contemplated that other agents may be used in combinationwith the present invention to improve the therapeutic efficacy oftreatment. One form of therapy for use in conjunction with chemotherapyincludes hyperthermia, which is a procedure in which a patient's tissueis exposed to high temperatures (up to 106° F.). External or internalheating devices may be involved in the application of local, regional,or whole-body hyperthermia. Local hyperthermia involves the applicationof heat to a small area, such as a tumor. Heat may be generatedexternally with high-frequency waves targeting a tumor from a deviceoutside the body. Internal heat may involve a sterile probe, includingthin, heated wires or hollow tubes filled with warm water, implantedmicrowave antennae, or radiofrequency electrodes.

[0205] A patient's organ or a limb is heated for regional therapy, whichis accomplished using devices that produce high energy, such as magnets.Alternatively, some of the patient's blood may be removed and heatedbefore being perfused into an area that will be internally heated.Whole-body heating may also be implemented in cases where cancer hasspread throughout the body. Warm-water blankets, hot wax, inductivecoils, and thermal chambers may be used for this purpose.

[0206] Hormonal therapy may also be used in conjunction with the presentinvention. The use of hormones may be employed in the treatment ofcertain cancers such as breast, prostate, ovarian, or cervical cancer tolower the level or block the effects of certain hormones such astestosterone or estrogen and this often reduces the risk of metastases.

[0207] F. Vectors that Express Bcl-2

[0208] Within certain embodiments, expression vectors are employed toexogenously express a Bcl-2 polypeptide product in cells, especiallylymphoid cells. Expression requires that appropriate signals be providedin the vectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

[0209] (i) Regulatory Elements

[0210] Throughout this application, the term “expression construct” ismeant to include any type of genetic construct containing a nucleic acidcoding for a gene product in which part or all of the nucleic acidencoding sequence is capable of being transcribed and translated into apolypeptide product. An “expression cassette” is defined as a nucleicacid encoding a gene product under transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrase“under transcriptional control” means that the promoter is in thecorrect location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

[0211] The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

[0212] At least one module in each promoter functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as the promoter forthe mammalian terminal deoxynucleotidyl transferase gene and thepromoter for the SV40 late genes, a discrete element overlying the startsite itself helps to fix the place of initiation.

[0213] Additional promoter elements regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the tk promoter, thespacing between promoter elements can be increased to 50 bp apart beforeactivity begins to decline. Depending on the promoter, it appears thatindividual elements can function either co-operatively or independentlyto activate transcription.

[0214] In certain embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter, the Rous sarcoma viruslong terminal repeat, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose.

[0215] By employing a promoter with well-known properties, the level andpattern of expression of the protein of interest following transfectionor transformation can be optimized. Further, selection of a promoterthat is regulated in response to specific physiologic signals can permitinducible expression of the gene product. Tables 2 and 3 list severalregulatory elements that may be employed, in the context of the presentinvention, to regulate the expression of the gene of interest. This listis not intended to be exhaustive of all the possible elements involvedin the promotion of gene expression but, merely, to be exemplarythereof.

[0216] Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins.

[0217] The basic distinction between enhancers and promoters isoperational. An enhancer region as a whole must be able to stimulatetranscription at a distance; this need not be true of a promoter regionor its component elements. On the other hand, a promoter must have oneor more elements that direct initiation of RNA synthesis at a particularsite and in a particular orientation, whereas enhancers lack thesespecificities. Promoters and enhancers are often overlapping andcontiguous, often seeming to have a very similar modular organization.

[0218] Below is a list of viral promoters, cellular promoters/enhancersand inducible promoters/enhancers that could be used in combination withthe nucleic acid encoding a gene of interest in an expression construct(Table 2 and Table 3). Additionally, any promoter/enhancer combination(as per the Eukaryotic Promoter Data Base EPDB) could also be used todrive expression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct. TABLE 2 Promoterand/or Enhancer Promoter/Enhancer References Immunoglobulin Heavy ChainBanerji et al., 1983; Gilles et al., 1983; Grosschedl et al, 1985;Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger et al.,1984; Kiledjian et al., 1988; Porton et al.; 1990 Immunoglobulin LightChain Queen et al., 1983; Picard et al., 1984 T-Cell Receptor Luria etal., 1987; Winoto et al., 1989; Redondo et al.; 1990 HLA DQ a and/or DQβ Sullivan et al., 1987 β-Interferon Goodbourn et al., 1986, Fujita etal., 1987; Goodbourn et al., 1988 Interleukin-2 Greene et al., 1989Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC ClassII 5 Koch et al., 1989 MHC Class II HLA-DRa Sherman et al., 1989 β-ActinKawamoto et al., 1988; Ng et al.; 1989 Muscle Creatine Kinase (MCK)Jaynes et al., 1988; Horlick et al., 1989; Johnson et al., 1989Prealbumin (Transthyretin) Costa et al., 1988 Elastase I Ornitz et al.,1987 Metallothionein (MTII) Karin et al., 1987; Culotta et al., 1989Collagenase Pinkert et al., 1987; Angel et al., 1987a Albumin Pinkert etal., 1987; Tronche et al., 1989, 1990 α-Fetoprotein Godbout et al.,1988; Campere et al., 1989 t-Globin Bodine et al., 1987; Perez-Stable etal., 1990 β-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-rasTriesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM) α₁ -AntitrypainLatimer et al., 1990 H2B (TH2B) Histone Hwang et al., 1990 Mouse and/orType I Collagen Ripe et al., 1989 Glucose-Regulated Proteins Chang etal., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 HumanSerum Amyloid A (SAA) Edbrooke et al., 1989 Troponin I (TN I) Yutzey etal., 1989 Platelet-Derived Growth Factor Pech et al., 1989 (PDGF)Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al.,1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herret al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al.,1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka etal., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villierset al., 1984; Hen et al., 1986; Satake et al., 1988; Campbell and/orVillarreal, 1988 Retroviruses Kriegler et al., 1982, 1983; Levinson etal., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986;Miksicek et al., 1986; Celander et al., 1987; Thiesen et al., 1988;Celander et al., 1988; Choi et al., 1988; Reisman et al., 1989 PapillomaVirus Campo et al., 1983; Lusky et al., 1983; Spandidos and/or Wilkie,1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al., 1987;Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et al.,1987; Spandau et al., 1988; Vannice et al., 1988 Human ImmunodeficiencyVirus Muesing et al., 1987; Hauber et al., 1988; Jakobovits et al.,1988; Feng et al., 1988; Takebe et al., 1988; Rosen et al., 1988;Berkhout et al., 1989; Laspia et al., 1989; Sharp et al., 1989; Braddocket al., 1989 Cytomegalovirus (CMV) Weber et al., 1984; Boshart et al.,1985; Foecking et al., 1986 Gibbon Ape Leukemia Virus Holbrook et al.,1987; Quinn et al., 1989

[0219] TABLE 3 Inducible Elements Element Inducer References MT IIPhorbol Ester (TFA) Palmiter et al., 1982; Haslinger Heavy metals etal., 1985; Searle et al., 1985; Stuart et al., 1985; Imagawa et al.,1987, Karin et al., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV(mouse mammary Glucocorticoids Huang et al., 1981; Lee et al., tumorvirus) 1981; Majors et al., 1983; Chandler et al., 1983; Ponta et al.,1985; Sakai et al., 1988 β-Interferon poly(rI)x Tavernier et al., 1983poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 Collagenase PhorbolEster (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel etal., 1987b CRP IL-6, IL-1 Ku & Mortensen, 1993 SAA IL-6, IL-1 Jiang etal., 1995 SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX GeneInterferon, Newcastle Hug et al., 1988 Disease Virus GRP78 Gene A23187Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989 VimentinSerum Rittling et al., 1989 MHC Class I Gene H-2κb Interferon Blanar etal., 1989 HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a, 1990bAntigen Proliferin Phorbol Ester-TPA Mordacq et al., 1989 Tumor NecrosisFactor TPA Hensel et al., 1989 Thyroid Stimulating Thyroid HormoneChatterjee et al., 1989 Hormone αGene

[0220] Where a cDNA insert is employed, one will typically desire toinclude a polyadenylation signal to effect proper polyadenylation of thegene transcript. The nature of the polyadenylation signal is notbelieved to be crucial to the successful practice of the invention, andany such sequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

[0221] (ii) Selectable Markers

[0222] In certain embodiments of the invention, the cells containnucleic acid constructs of the present invention, a cell may beidentified in vitro or in vivo by including a marker in the expressionconstruct. Such markers would confer an identifiable change to the cellpermitting easy identification of cells containing the expressionconstruct. Usually the inclusion of a drug selection marker aids incloning and in the selection of transformants, for example, genes thatconfer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocinand histidinol are useful selectable markers. Alternatively, enzymessuch as herpes simplex virus thymidine kinase (tk) or chloramphenicolacetyltransferase (CAT) may be employed. Immunologic markers also can beemployed. The selectable marker employed is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable markers are well known to one of skill in the art.

[0223] (iii) Polyadenylation Signals

[0224] In expression, one will typically include a polyadenylationsignal to effect proper polyadenylation of the transcript. The nature ofthe polyadenylation signal is not believed to be crucial to thesuccessful practice of the invention, and/or any such sequence may beemployed. Preferred embodiments include the SV40 polyadenylation signaland/or the bovine growth hormone polyadenylation signal, convenientand/or known to function well in various target cells. Also contemplatedas an element of the expression cassette is a transcriptionaltermination site. These elements can serve to enhance message levelsand/or to minimize read through from the cassette into other sequences.

[0225] (iv) Vectors

[0226] The term “vector” is used to refer to a carrier nucleic acidmolecule into which a nucleic acid sequence can be inserted forintroduction into a cell where it can be replicated. A nucleic acidsequence can be “exogenous,” which means that it is foreign to the cellinto which the vector is being introduced or that the sequence ishomologous to a sequence in the cell but in a position within the hostcell nucleic acid in which the sequence is ordinarily not found. Thus,in the present invention a Bcl-2 from any cell may be expressed in alymphoid cell that normally does not express Bcl-2. Vectors includeplasmids, cosmids, viruses (bacteriophage, animal viruses, and plantviruses), and artificial chromosomes (e.g., YACs). One of skill in theart would be well equipped to construct a vector through standardrecombinant techniques, which are described in Sambrook et al. (1989)and Ausubel et al. (1994), both incorporated herein by reference.

[0227] The term “expression vector” refers to a vector containing anucleic acid sequence coding for at least part of a gene product capableof being transcribed. In some cases, RNA molecules are then translatedinto a protein, polypeptide, or peptide. In other cases, these sequencesare not translated, for example, in the production of antisensemolecules or ribozymes. Expression vectors can contain a variety of“control sequences,” which refer to nucleic acid sequences necessary forthe transcription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

[0228] (v) Delivery of Expression Vectors

[0229] There are a number of ways in which expression vectors may beintroduced into cells. In certain embodiments of the invention, theexpression construct comprises a virus or engineered construct derivedfrom a viral genome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden,1986; Temin, 1986). The first viruses used as gene vectors were DNAviruses including the papovaviruses (simian virus 40, bovine papillomavirus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) andadenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have arelatively low capacity for foreign DNA sequences and have a restrictedhost spectrum. Furthermore, their oncogenic potential and cytopathiceffects in permissive cells raise safety concerns. They can accommodateonly up to 8 kB of foreign genetic material but can be readilyintroduced in a variety of cell lines and laboratory animals (Nicolasand Rubenstein, 1988; Temin, 1986).

[0230] Adenovirus. One of the methods for in vivo delivery involves theuse of an adenovirus expression vector. “Adenovirus expression vector”is meant to include those constructs containing adenovirus sequencessufficient to (a) support packaging of the construct and (b) to expressan antisense polynucleotide that has been cloned therein. In thiscontext, expression does not require that the gene product besynthesized.

[0231] The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization of adenovirus, a 36kB, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and no genome rearrangementhas been detected after extensive amplification. Adenovirus can infectvirtually all epithelial cells regardless of their cell cycle stage. Sofar, adenoviral infection appears to be linked only to mild disease suchas acute respiratory disease in humans.

[0232] Adenovirus is particularly suitable for use as a gene transfervector because of its mid-sized genome, ease of manipulation, hightiter, wide target cell range and high infectivity. Both ends of theviral genome contain 100-200 base pair inverted repeats (ITRs), whichare cis elements necessary for viral DNA replication and packaging. Theearly (E) and late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and a fewcellular genes. The expression of the E2 region (E2A and E2B) results inthe synthesis of the proteins for viral DNA replication. These proteinsare involved in DNA replication, late gene expression and host cellshut-off (Renan, 1990). The products of the late genes, including themajority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and all themRNA's issued from this promoter possess a 5′-tripartite leader (TPL)sequence which makes them preferred mRNA's for translation.

[0233] In a current system, recombinant adenovirus is generated fromhomologous recombination between shuttle vector and provirus vector. Dueto the possible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and examineits genomic structure.

[0234] Generation and propagation of the current adenovirus vectors,which are replication deficient, depend on a unique helper cell line,designated 293, which was transformed from human embryonic kidney cellsby Ad5 DNA fragments and constitutively expresses E1 proteins (Graham etal., 1977). Since the E3 region is dispensable from the adenovirusgenome (Jones and Shenk, 1978), the current adenovirus vectors, with thehelp of 293 cells, carry foreign DNA in either the E1, the D3 or bothregions (Graham and Prevec, 1991). In nature, adenovirus can packageapproximately 105% of the wild-type genome (Ghosh-Choudhury et al.,1987), providing capacity for about 2 extra kb of DNA. Combined with theapproximately 5.5 kb of DNA that is replaceable in the E1 and E3regions, the maximum capacity of the current adenovirus vector is under7.5 kb, or about 15% of the total length of the vector. More than 80% ofthe adenovirus viral genome remains in the vector backbone and is thesource of vector-borne cytotoxicity. Also, the replication deficiency ofthe E1-deleted virus is incomplete. For example, leakage of viral geneexpression has been observed with the currently available vectors athigh multiplicities of infection (MOI) (Mulligan, 1993).

[0235] Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. As stated above,the preferred helper cell line is 293.

[0236] Racher et al. (1995) disclosed improved methods for culturing 293cells and propagating adenovirus. In one format, natural cell aggregatesare grown by inoculating individual cells into 1 liter siliconizedspinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium.Following stirring at 40 rpm, the cell viability is estimated withtrypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin,Stone, UK) (5 g/l) is employed as follows. A cell innoculum, resuspendedin 5 ml of medium, is added to the carrier (50 ml) in a 250 mlErlenmeyer flask and left stationary, with occasional agitation, for 1to 4 h. The medium is then replaced with 50 ml of fresh medium andshaking initiated. For virus production, cells are allowed to grow toabout 80% confluence, after which time the medium is replaced (to 25% ofthe final volume) and adenovirus added at an MOI of 0.05. Cultures areleft stationary overnight, following which the volume is increased to100% and shaking commenced for another 72h.

[0237] Other than the requirement that the adenovirus vector bereplication defective, or at least conditionally defective, the natureof the adenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

[0238] As stated above, the typical vector according to the presentinvention is replication defective and will not have an adenovirus E1region. Thus, it will be most convenient to introduce the polynucleotideencoding the gene of interest at the position from which the E1-codingsequences have been removed. However, the position of insertion of theconstruct within the adenovirus sequences is not critical to theinvention. The polynucleotide encoding the gene of interest may also beinserted in lieu of the deleted E3 region in E3 replacement vectors, asdescribed by Karlsson et al. (1986), or in the E4 region where a helpercell line or helper virus complements the E4 defect.

[0239] Adenovirus is easy to grow and manipulate and exhibits broad hostrange in vitro and in vivo. This group of viruses can be obtained inhigh titers, e.g., 09-1012 plaque-forming units per ml, and they arehighly infective. The life cycle of adenovirus does not requireintegration into the host cell genome. The foreign genes delivered byadenovirus vectors are episomal and, therefore, have low genotoxicity tohost cells. No side effects have been reported in studies of vaccinationwith wild-type adenovirus (Couch et al., 1963; Top et al., 1971),demonstrating their safety and therapeutic potential as in vivo genetransfer vectors.

[0240] Adenovirus vectors have been used in eukaryotic gene expression(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development(Grunhaus & Horwitz, 1992; Graham and Prevec, 1991). Recently, animalstudies suggested that recombinant adenovirus could be used for genetherapy (Stratford-Perricaudet & Perricaudet, 1991;Stratford-Perricaudet et al., 1990; Rich et al., 1993). Studies inadministering recombinant adenovirus to different tissues includetrachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992),muscle injection (Ragot et al., 1993), peripheral intravenous injections(Herz and Gerard, 1993) and stereotactic inoculation into the brain (LeGal La Salle et al., 1993).

[0241] Retrovirus. The retroviruses are a group of single-stranded RNAviruses characterized by an ability to convert their RNA todouble-stranded DNA in infected cells by a process ofreverse-transcription (Coffin, 1990). The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Thesecontain strong promoter and enhancer sequences and are also required forintegration in the host cell genome (Coffin, 1990).

[0242] In order to construct a retroviral vector, a nucleic acidencoding a gene of interest is inserted into the viral genome in theplace of certain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and stable expressionrequire the division of host cells (Paskind et al., 1975).

[0243] A novel approach designed to allow specific targeting ofretrovirus vectors was recently developed based on the chemicalmodification of a retrovirus by the chemical addition of lactoseresidues to the viral envelope. This modification could permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

[0244] A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, they demonstrated the infection of avariety of human cells that bore those surface antigens with anecotropic virus in vitro (Roux et al., 1989).

[0245] There are certain limitations to the use of retrovirus vectors inall aspects of the present invention. For example, retrovirus vectorsusually integrate into random sites in the cell genome. This can lead toinsertional mutagenesis through the interruption of host genes orthrough the insertion of viral regulatory sequences that can interferewith the function of flanking genes (Varmus et al., 1981). Anotherconcern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which theintact-sequence from the recombinant virus inserts upstream from thegag, pol, env sequence integrated in the host cell genome. However, newpackaging cell lines are now available that should greatly decrease thelikelihood of recombination (Markowitz et al., 1988; Hersdorffer et al.,1990).

[0246] Adeno-Associated Viruses. Adeno-associated virus (AAV) is anattractive virus for delivering foreign genes to mammalian subjects(Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska,1984). AAV utilizes a linear, single-stranded DNA of about 4700 basepairs. Inverted terminal repeats flank the genome. Two genes are presentwithin the genome, giving rise to a number of distinct gene products.The first, the cap gene, produces three different virion proteins (VP),designated VP-1, VP-2 and VP-3. The second, the rep gene, encodes fournon-structural proteins (NS). One or more of these rep gene products isresponsible for transactivating AAV transcription. The sequence of AAVis provided by U.S. Pat. No. 5,252,479 (entire text of which isspecifically incorporated herein by reference).

[0247] The three promoters in AAV are designated by their location, inmap units, in the genome. These are, from left to right, p5, p19 andp40. Transcription gives rise to six transcripts, two initiated at eachof three promoters, with one of each pair being spliced. The splicesite, derived from map units 42-46, is the same for each transcript. Thefour non-structural proteins apparently are derived from the longer ofthe transcripts, and three virion proteins all arise from the smallesttranscript.

[0248] AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

[0249] The terminal repeats of the AAV vector of the present inventioncan be obtained by restriction endonuclease digestion of AAV or aplasmid such as p201, which contains a modified AAV genome (Samulski etal., 1987). Alternatively, the terminal repeats may be obtained by othermethods known to the skilled artisan, including but not limited tochemical or enzymatic synthesis of the terminal repeats based upon thepublished sequence of AAV. The ordinarily skilled artisan can determine,by well-known methods such as deletion analysis, the minimum sequence orpart of the AAV ITRs which is required to allow function, i.e., stableand site-specific integration. The ordinarily skilled artisan also candetermine which minor modifications of the sequence can be toleratedwhile maintaining the ability of the terminal repeats to direct stable,site-specific integration.

[0250] Other Viruses. Other viral vectors may be employed as expressionconstructs in the present invention. Vectors derived from viruses suchas vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988) and herpesviruses may be employed. They offer severalattractive features for various mammalian cells (Friedmann, 1989;Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwichet al., 1990).

[0251] With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. The hepatotropism and persistence(integration) were particularly attractive properties for liver-directedgene transfer. Chang et al. recently introduced the chloramphenicolacetyltransferase (CAT) gene into duck hepatitis B virus genome in theplace of the polymerase, surface, and pre-surface coding sequences. Itwas co-transfected with wild-type virus into an avian hepatoma cellline. Culture media containing high titers of the recombinant virus wereused to infect primary duckling hepatocytes. Stable CAT gene expressionwas detected for at least 24 days after transfection (Chang et al.,1991).

[0252] Non-Viral Methods. Several non-viral methods for the transfer ofexpression constructs into mammalian cells also are contemplated by thepresent invention. These include DEAE-dextran (Gopal, 1985),electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), directmicroinjection (Harland and Weintraub, 1985), lipofectamine-DNAcomplexes, cell sonication (Fechheimer et al., 1987), andreceptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).

[0253] In yet another embodiment of the invention, the expressionconstruct may simply consist of naked recombinant DNA or plasmids.Transfer of the construct may be performed by any of the methodsmentioned above which physically or chemically permeabilize the cellmembrane. This is particularly applicable for transfer in vitro but itmay be applied to in vivo use as well. Dubensky et al. (1984)successfully injected polyomavirus DNA in the form of calcium phosphateprecipitates into liver and spleen of adult and newborn micedemonstrating active viral replication and acute infection. Benvenistyand Neshif (1986) also demonstrated that direct intraperitonealinjection of calcium phosphate-precipitated plasmids results inexpression of the transfected genes. It is envisioned that DNA encodinga gene of interest may also be transferred in a similar manner in vivoand express the gene product.

[0254] Liposomes. In a further embodiment of the invention, theexpression construct may be entrapped in a liposome. Liposomes arevesicular structures characterized by a phospholipid bilayer membraneand an inner aqueous medium. Multilamellar liposomes have multiple lipidlayers separated by aqueous medium. They form spontaneously whenphospholipids are suspended in an excess of aqueous solution. The lipidcomponents undergo self-rearrangement before the formation of closedstructures and entrap water and dissolved solutes between the lipidbilayers (Ghosh and Bachhawat, 1991). Also contemplated areLipofectamine®-DNA complexes.

[0255] Liposome-mediated nucleic acid delivery and expression of foreignDNA in vitro has been very successful. Wong et al. (1980) demonstratedthe feasibility of liposome-mediated delivery and expression of foreignDNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al.(1987) accomplished successful liposome-mediated gene transfer in ratsafter intravenous injection.

[0256] In certain embodiments of the invention, the liposome may becomplexed with a hemagglutinating virus (HVJ). This has been shown tofacilitate fusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

[0257] Other expression constructs which can be employed to deliver anucleic acid encoding a particular gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

[0258] Receptor-mediated gene targeting vehicles generally consist oftwo components: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferrin (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0 273 085).

[0259] In other embodiments, the delivery vehicle may comprise a ligandand a liposome. For example, Nicolau et al. (1987) employedlactosyl-ceramide, a galactose-terminal asialganglioside, incorporatedinto liposomes and observed an increase in the uptake of the insulingene by hepatocytes. Thus, it is feasible that a nucleic acid encoding aparticular gene also may be specifically delivered into a cell type byany number of receptor-ligand systems with or without liposomes. Forexample, epidermal growth factor (EGF) may be used as the receptor formediated delivery of a nucleic acid into cells that exhibit upregulationof EGF receptor. Mannose can be used to target the mannose receptor onliver cells.

[0260] G. Pharmaceutical Formulations and Delivery

[0261] In a preferred embodiment of the present invention, a method oftreatment for a cancer by administering to a cancer cell CDDO-compoundand a chemotherapeutic agent, wherein the combination of theCDDO-compound with the chemotherapeutic agent is effective in inducingcytotoxicity in said cell

[0262] An effective amount of the pharmaceutical composition, generally,is defined as that amount sufficient to detectably and repeatedly toameliorate, reduce, minimize or limit the extent of the disease or itssymptoms. More rigorous definitions may apply, including elimination,eradication or cure of disease.

[0263] The routes of administration will vary, naturally, with thelocation and nature of the lesion, and include, e.g., intradermal,transdermal, parenteral, intravenous, intra-arterial, intramuscular,intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal,intratumoral, perfusion, lavage, direct injection, ex vivo bone marrowor blood cell purging, and oral administration and formulation.Intratumoral injection, or injection into the tumor vasculature isspecifically contemplated for discrete, solid, accessible tumors. Local,regional or systemic administration also may be appropriate. In the caseof surgical intervention, the present invention may be used beforesurgery, at the time of surgery, and/or thereafter, to treat residual ormetastatic disease. For example, a resected tumor bed may be injected orperfused with a formulation comprising the combination of theCDDO-compound and other chemotherapeutic therapy of this invention. Theperfusion may be continued post-resection, for example, by leaving acatheter implanted at the site of the surgery. Periodic post-surgicaltreatment also is envisioned. Ex vivo purging is an important method ofadministration that is contemplated.

[0264] Continuous administration also may be applied where appropriate,for example, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery may be via syringe orcatherization. Such continuous perfusion may take place for a periodfrom about 1-2 hours, to about 2-6 hours, to about 6-12 hours, to about12-24 hours, to about 1-2 days, to about 1-2 wk or longer following theinitiation of treatment. Generally, the dose of the therapeuticcomposition via continuous perfusion will be equivalent to that given bya single or multiple injections, adjusted over a period of time duringwhich the perfusion occurs. It is further contemplated that limbperfusion may be used to administer therapeutic compositions of thepresent invention, particularly in the treatment of melanomas andsarcomas.

[0265] Treatment regimens may vary as well, and often depend on tumortype, tumor location, disease progression, and health and age of thepatient. Obviously, certain types of tumors will require more aggressivetreatment, while at the same time, certain patients cannot tolerate moretaxing protocols. The clinician will be best suited to make suchdecisions based on the known efficacy and toxicity (if any) of thetherapeutic formulations.

[0266] Solutions of the active compounds as free base orpharmacologically acceptable salts may be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions mayalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy syringability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

[0267] For parenteral administration in an aqueous solution, forexample, the solution should be suitably buffered if necessary and theliquid diluent first rendered isotonic with sufficient saline orglucose. These particular aqueous solutions are especially suitable forintravenous, intramuscular, subcutaneous, intratumoral andintraperitoneal administration. In this connection, sterile aqueousmedia that can be employed will be known to those of skill in the art inlight of the present disclosure. For example, one dosage may bedissolved in 1 ml of isotonic NaCl solution and either added to 1000 mlof hypodermoclysis fluid or injected at the proposed site of infusion,(see for example, “Remington's Pharmaceutical Sciences” 15th Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

[0268] Sterile injectable solutions are prepared by incorporating theactive compounds in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vaccuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0269] The compositions disclosed herein may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as injectable solutions, drug releasecapsules and the like.

[0270] As used herein, “carrier” includes any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

[0271] The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared.

[0272] H. Clinical Trials

[0273] This section is concerned with the development of human treatmentprotocols for anticancer therapy using the CDDO-compounds in combinationwith other chemotherapeutic agents and/or immunosuppressive agents.

[0274] The various elements of conducting a clinical trial, includingpatient treatment and monitoring, will be known to those of skill in theart in light of the present disclosure. The following information isbeing presented as a general guideline for use in establishing theCDDO-compound based combination therapies described herein alone or incombinations with other adjunct treatments used routinely in cancertherapy in clinical trials.

[0275] Candidates for the phase 1 clinical trial will be patients onwhich all conventional therapies have failed. Approximately 100 patientswill be treated initially. Their age will range from 16 to 90 (median65) years. Patients will be treated, and. samples obtained, without biasto sex, race, or ethnic group. For this patient population ofapproximately 41% will be women, 6% will be black, 13% Hispanic, and 3%other minorities. These estimates are based on consecutive cases seen atMD Anderson Cancer Center over the last 5 years.

[0276] Optimally the patient will exhibit adequate bone marrow function(defined as peripheral absolute granulocyte count of >2,000/mm³ andplatelet count of 100,000/mm³, adequate liver function (bilirubin 1.5mg/dl) and adequate renal function (creatinine 1.5 mg/dl).

[0277] Research samples will be obtained from peripheral blood or marrowunder existing IRB approved projects and protocols. Some of the researchmaterial will be obtained from specimens taken as part of patient care.

[0278] The subacute and chronic toxicity studies, pharmacokinetics andtissue distribution studies (described in the section entitled Examples)provide the critical information necessary to generate and have approvedan Investigational New Drug (IND) for the CDDO-compound-chemotherapeuticagent combination therapy described in this invention for clinicalstudies in leukemia and other cancer patients.

[0279] The combination treatments described above will be administeredto the patients regionally or systemically on a tentative weekly basis.A typical treatment course may comprise about six doses delivered over a7 to 21 day period. Upon election by the clinician the regimen may becontinued with six doses every three weeks or on a less frequent(monthly, bimonthly, quarterly etc.) basis. Of course, these are onlyexemplary times for treatment, and the skilled practitioner will readilyrecognize that many other time-courses are possible.

[0280] The modes of administration may be local administration,including, by intratumoral injection and/or by injection into tumorvasculature, intratracheal, endoscopic, subcutaneos, and/orpercutaneous. The mode of administration may be systemic, including,intravenous, intra-arterial, intra-peritoneal and/or oraladministration.

[0281] In one embodiment, CDDO and retinoids will be administered. Inother embodiments, CDDO-Me and retinoids will be administered.

[0282] CDDO will be administered at dosages in the range of 5-30 mg/kgintravenously or 5-100 mg/kg orally. CDDO-Me will be administered in therange of 5-100 mg/kg intravenously or 5-100 mg/kg orally for 3-30 days.The retinoid may be all-trans-retinoic acid (ATRA), 9-cis-retinoic acid,LG100268, LGD1069 (Targretin, bexarotene), fenretinide[N-(4-hydroxyphenyl)retinamide, 4-HPR], CD437 or any RXR- orRAR-specific retinoic acid. The retionids may be administered orally,intravenously, by topical application or by other routes. In someembodiments the retinoids are liposomal formulations. For example, aliposomal formulation of ATRA is administered a range of 10-100mg/m²/day intravenously. Thus, one may administer to a patient 10, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100mg/m²/day of a liposomal formulation of ATRA. Non-liposomal ATRA may beadministered orally in the range of 10-100 mg/m²/day. Thus, one mayadminister to a patient 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100 mg/m²/day of ATRA orally. 9-cis-Retinoid acidmay be administered in the range of 20-150 mg/m² twice a day orally.Thus, one may administer 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150mg/m² of 9-cis-retinoid. LG100268 is be effective in a dose range of5-50 mg/kg. Thus, 5, 10, 15, 20, 25, 30, 35, 40, 45, to 50 mg/kg ofLG100268 may be administered to a patient. LGD1069 (Targretin,bexarotene) capsules are contemplated for the topical treatment ofcutaneous lesions in patients with cutaneous T-cell lymphoma (CTCL) whohave refractory or resistant disease after other therapies. The doseranges of these capusles is 300-400 mg/m²/day orally. LGD1069 gel at 1%may also be used for the topical treatment of cutaneous lesions inpatients with CTCL (Stage (1A and 1B) who have refractory or resistantdisease after other therapies; two to four times daily. Fenretinide[N-(4-hydroxyphenyl)retinamide, 4-HPR] is contemplated useful at 25-600mg daily and the administration in some embodiments may be continuous.Of course, the skilled artisan will understand that while these dosageranges, provide useful guidelines appropriate adjustments in the dosagedepending on the needs of an individual patient factoring in disease,gender, age and other general health conditions will be made at the timeof administration to a patient by a trained physician. The same is truefor means of adminitration, routes of administration as well.

[0283] In yet other embodiments, CDDO-compounds and otherchemotherapeutic drugs such as Doxorubicin, Mylotarg, Dolastatin-10,Bryostatin or any other chemotherapeutic drug used in cancer therapywill be administered to patients in need thereof. Some exemplary dosesand routes of various chemotherapeutics are listed here: Taxol isusually administered by IV at one dose of 130-250 mg/m² every 3 weeks,Vincristine is usually administered by IV at one dose of 1-1.4 mg/m²every week, Vinblastine is usually given by IV at one dose of 6 mg/m²every week, or at a dose of 2 mg/m² as a continuous infusion for 5 daysevery 3 weeks, VP-16 (etoposide) is usually administered by IV at 75-100mg/m² every day for 5 days, or orally at a dose of 200 mg/m² for 2 daysevery week, Actinomycin D is usually administered by IV at one dose of0.6 mg/m² every day for 5 days, Doxorubicin is typically administered byIV at one dose of 75 mg/m² every 3 weeks, or at a dose of 20 mg/m² everyweek, Daunorubicin is usually administered by IV at a dose of 30 mg/m²for 3 days every 3 weeks, liposomal Daunorubicin 40-100 mg/m² every dayfor 3 days, Idarubicin is usually administered by IV at a dose of 13mg/m² every day for 3 days, Mitomycin-C is usually administered by IV ata dose of 10 mg/m² every day for 3 days, and is repeated every 3 weeks,Actinomycin D IV 0.3-06 mg/m² every day for 5 days, Bleomycin can beadministered by IV, IM, or SC at one dose of 2-15 mg/m² every week,Methrotrexate may be administered by IV or IM at a dose of 25 mg/m²twice weekly, it may also be administered in a high dose of >500 mg/m²every 3 weeks in conjunction with IV leucovorin at a dose of 15 mg/m²every 6 hours for 7 doses, Cisplatin is typically administered IV 20-40mg/m² every day for 5 days, or IV 50-100 mg/m² every 3-4 weeks, Taxol IV130-250 mg/m² every 3 weeks, or 750 mg/m² every 3 weeks, 5-Fluorouracilcan be administered by IV at a dose of 500 mg/m² every week or for 5days every 4 weeks, it can also be administered by IV at a dose of800-1200 mg/m² every 3-4 weeks. Another administration method isintraarterial (IA) at a dose of 800-1200 mg/m² every day for 14-21 days.5-Fluorouracil can also be administered IV at a dose of 375-600 mg/m²once every week for 6 weeks in conjunction with IV leucovorin at a doseof 500 mg/m² once every week for 6 weeks. Cytarabine can be administeredby IV at a dose of 100 mg/m² every 12 hours for 5-10 days or bycontinuous infusion. Hydroxyurea can be administered by IV at a dose of1000-1500 mg/m² every day for 5 days or orally at a dose of 1000 mg/m²every day. Fludarabine can be administered by IV at a dose of 25 mg/m²every day for 5 days. Cyclophosphamide IV 400 mg/m² bolus every day for5 days; Orally 100-300 mg every day for 14 days. Carmusitin (BCNU)200-225 mg/m² once every 6 weeks. Melphalan IV 8 mg/m² every day for 5days; orally 4 mg/m² daily. Chlorambucil orally 1-3 mg/m² daily.Busulfan orally 2-6 mg/m² daily. Lomustine (CCNU) orally 100-150 mg/m²once every 6 weeks. TRAIL is another biotherapeutic agent that may beused in conjuction with the CDDO-compounds presented herein. TRAIL is amember of tumor necrosis factor family of cytokines. Trials in humansare underway, so the exact MTD is not known at this point. In mice,daily injections of 200-1000 μg of TRAIL (14 days) significantlyincreased survival of tumor-bearing mice (see Walczak et al., 1999).Combination of 250-500 μg of TRAIL synergistically enhanced effect ofchemotherapy in mice bearing human colon carcinoma tumors (Gliniak etal., 1999). Thus, the inventors contemplate using Dolastatin-10 IV300-2000 μg/m² every 3 weeks. Bryostatin IV 12.5-50 μg/m² every 2 weeks.Liposomal Annamycin IV 100-350 mg/m² every day for 3 days. Mylotarg IV4.5-9 mg/m² every 4 weeks. The present inventors also contemplate usingchemotherapeutics that are differentiating agents such as SodiumPhenylacetate (NAPA) at a dose of 200-600 mg/kg/day IV continuousinfusion for 14 days and/or Sodium Butyrate (NAPB)-500-2000 mg/kg/dayfor 7 days IV continuous infusion, SAHA or other histone deacetylaseinhibitors.

[0284] A description of dosage ranges and routes of administration forother chemotherapeutics is also presented in the section on cancertherapies. It will be understood that the exact dose, frequency androute of administration of the combination therapies of this inventionwill be determined by one of skill in the art taking into account theage, sex, type of cancer, and other health factors of the individualpatient.

[0285] To monitor disease course and evaluate the cancer cell killing itis contemplated that the patients should be examined for appropriatetests every month. To assess the effectiveness of the drug, thephysician will determine parameters to be monitored depending on thetype of cancer/tumor and will involve methods to monitor reduction intumor mass by for example computer tomography (CT) scans, detection ofthe presense of the PSA (prostrate specific antigen) in prostratecancer, HCG in germ tumor and the like. Tests that will be used tomonitor the progress of the patients and the effectiveness of thetreatments include: physical exam, X-ray, blood work, bone marrow workand other clinical laboratory methodologies. The doses given in thephase 1 study will be escalated as is done in standard phase 1 clinicalphase trials, i.e., doses will be escalated until maximal tolerableranges are reached.

[0286] Clinical responses may be defined by acceptable measure. Forexample, a complete response may be defined by complete disappearance ofthe leukemia or cancer cells, whereas a partial response may be definedby a 50% reduction of leukemia or cancer cells.

[0287] The typical course of treatment will vary depending upon theindividual patient and disease being treated in ways known to those ofskill in the art. For example, a patient with AML might be treated infour week cycles, although longer duration may be used if no adverseeffects are observed with the patient, and shorter terms of treatmentmay result if the patient does not tolerate the treatment as hoped. Thistreatment may be repeated for 6-24 months.

I. EXAMPLES

[0288] The following examples are included to demonstrate preferredembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

Example 1 Materials and Methods

[0289] Reagents. Stock solutions of CDDO and/or CDDO-Me at 10 mM in DMSOwere stored at −20° C. Working solutions were prepared in DMSO and addeddirectly to culture medium. ATRA was purchased from Sigma Chemical Co.(St. Louis, Mo.) and kept in 100% ethanol solution at −20° C.RXR-specific ligand LG-100268 was kindly provided by Dr. Richard Heyman.Caspase-3 inhibitor Z-DEVD-fmk and bongkrekic acid (BA) were obtainedfrom Calbiochem (La Jolla, Calif.). Cyclosporin A (CyA) was purchasedfrom Sandoz. Fas-signaling antibody CH11 and Fas-blocking antibody ZB4were obtained from Immunotech (Miami, Fla.).

[0290] Cell lines. HL-60, KG-1, U937, and Jurkat cell lines wereobtained from the American Type Culture Collection (Rockville, Md.). NB4cells were kindly provided by Dr. M. Lanotte.HL-60-doxorubicin-resistant cells (HL-60-DOX) were also used. U937/Bcl-2and its appropriate vector controls (U937/pCEP) were provided by Dr. S.Grant. U937 cells were transfected with WT, S70A, or S70E cDNAcontaining cytomegalovirus plasmids by electroporation (200 V, 975 μFcapacitance) and selected and maintained in the medium plus 500 μg/mLG418 (Gibco BRL, Gaithersburg, Md.)

[0291] Subjects. Samples of bone marrow or peripheral blood wereobtained for in vitro studies from patients with newly diagnosed orrecurrent AML with high (>70%) blast count and from patients withmyeloid transformation of chronic myeloid leukemia (CML). Informedconsent was obtained following institutional guidelines. Mononuclearcells were separated by Ficoll-Hypaque (Sigma Chemical Co)density-gradient centrifugation.

[0292] Suspension culture of leukemic cells. Leukemic cell lines werecultured at a density of 3.0×10⁵ cells/mL, and AML mononuclear cells at5×10⁵ cells/mL in the presence or absence of indicated concentrations ofCDDO-Me. Appropriate amounts of DMSO (final concentration <0.05%) wasincluded as a control. For cytotoxicity studies, 1 μM ara-C was added tothe cultures. After 24-72 hours, viable cells were counted with theTrypan blue-dye exclusion method using a hematocytometer.

[0293] Cell kinetic and DNA fragmentation studies. The cell cyclekinetics was determined by staining cells with acridine orange forcellular DNA and RNA content followed by flow cytometeric analysis asdescribed. Samples were measured in a FACScan flow cytometer (BectonDickinson, San Jose, Calif.) using the 488-nm line of a 15-nm argonlaser and filter settings for green (530 nm) (DNA) and red (585 nm)(RNA) fluorescence. Ten thousand events were stored in list mode foranalysis. The percentage of cells in the “sub G₁ peak” defined theproportion of apoptotic cells in the tested populations. Cell debris wasdefined as events in the lowest 10% range of fluorescence, and theseresults were eliminated from analysis. Cell-cycle kinetics was analyzedusing ModFit software (Verity Software House, Inc., Topsham, Me.).

[0294] AML blast colony assay. A previously described method was used tomeasure AML blast colony formation. Briefly, 1×10⁵ T-cell-depleted,nonadherent, low-density bone marrow cells were plated in 0.8%methylcellulose in Iscove's modified Dulbecco's medium (IMDM; GibcoLaboratories, Grand Island, N.Y.) supplemented with 10% fetal bovineserum and 15 ng/mL recombinant human granulocyte-macrophagecolony-stimulating factor (hGM-CSF). CDDO or CDDO-Me was added at theinitiation of cultures at concentrations ranging from 0.05 to 0.5 μg/mL.AML blast colonies were evaluated under a microscope on day 7 of culturein duplicate dishes.

[0295] CFU-Granulocyte-Erythroid-Macrophage-Megakaryocyte (CFU-GEMM)Assay. In three experiments, 2×10⁵ CD34+cells isolated from normal bonemarrow (n=1) or G-CSF-stimulated peripheral blood (n=2) were plated in0.8% methylcellulose with IMDM, 1 U/mL human erythropoietin (Terry FoxLaboratories, Vancouver, Canada), and 50 ng/mL recombinant hGM-CSF.CDDO-Me was added at the initiation of cultures at concentrationsranging from 0.05 to 0.5 μg/mL. All cultures were evaluated after 14days for the number of burst forming unit-erythroid (BFU-E) colonies,defined as aggregate of more than 500 hemoglobinized cells or three ormore erythroid subcolonies and CFU-GM colonies, defined as a cluster of40 or more granulocytes, monocyte-macrophages or both.

[0296] Western blot analysis. An equal amount of protein lysate wasplaced on 12% SDS-PAGE for 2 hours at 100 volts, followed by transfer ofthe protein to a Nytran membrane (S&S, Heween, N.H.) and immunoblotting.Polyclonal rabbit antibodies to Bcl-2, Bcl-X_(L), and Bax were kindlyprovided by Dr. J. C. Reed. Antibodies against PARP was obtained fromPharMingen (San Diego, Calif.), DFF-45 from Oncogene (Cambridge, Mass.),XIAP from Transduction Laboratories (Lexington, Ky.), caspase-3 fromPharMingen, phospho-specific anti-pERK1/2 antibodies from Calbiochem(San Diego, Calif.). A specific antibody recognizing only thep20-processed caspase-3 band was provided by Idun Pharmaceutical, Inc.(La Jolla, Calif.).

[0297] Cell fractionation and Bax immunolocalization studies. Thesubcellular fractionation of cells was performed by a previouslydescribed method. Briefly, cells were swollen in ice-cold hypotonicHepes buffer (10 mM Hepes at pH 7.4/5 mM MgCl₂/40 mM KCl/1 mM PMSF/10μg/mL aprotinin/10 μg/mL leupeptin) for 30 minutes, aspirated repeatedlythrough a 25-gauge needle (25 strokes), and centrifuged at 200× g topellet the nuclei. The resulting supernatant was then centrifuged at10,000 x g to pellet the heavy-membrane (HM) fraction containing themitochondria. The HM supernatant was centrifuged at 150,000× g to pelletthe plasma membranes, and the supernatant represented the cytosol (Cyt).Subcellular fractions were subjected to denaturing electrophoresis in a12% acrylamide/0.1% SDS gel and transferred to nitrocellulose for Baxwestern blotting.

[0298] Northern blot analysis. The Bax probe was obtained by cloning thepolymerase chain reaction (PCR) products of amplified cDNA. The sequencewas compared with Genbank data to ensure that the correct cDNA wascloned. Twenty micrograms of total RNA was denatured and run overnighton a 1% formamide agarose gel at 30 volts. After staining in ethidiumbromide, RNA was transferred to a Nitran filter and left overnight in10× sodium chloride/sodium citrate (SSC), followed by drying at 80° C.Hybridization was carried out at 42° C. for 20 hours, and the filterswere washed under highly stringent conditions. Signals were analyzedwith a Betascope 603 (Betagen, Waltham, Mass.).

[0299] Metabolic labeling, immunoprecipitation, and immunoblot analysis.Cells were labeled with [³²P]orthophosphoric acid and then treated with0.1 μM CDDO-Me or CDDO, after which Bcl-2 was analyzed byimmunoprecipitation, as previously described Samples wereelectrophoresed in a 12% acrylamide/0.1% SDS gel, transferred tonitrocellulose, and exposed to Hyperfilm (Amersham Pharmacia Biotech,UK) at −80° C. The same blot was used for western blot analysis withanti-Bcl-2 antisera.

[0300] In vitro ERK assay. The effect of CDDO or CDDO-Me was determinedusing an in vitro MAPK kinase assay kit from Upstate Biotechnology (LakePlacid, N.Y.) and ERK1/2 antibody from Santa Cruz Biotechnology (SantaCruz, Calif.). For each sample, ERK 1/2 was immunoprecipitated from2×10⁷ K562 or 1×10⁷ HL60 cells using a specific anti-ERK 1/2 antibodyand Protein A agarose (Life Technologies, Rockville, Md.). TheERK-containing agarose pellet was resuspended in Assay Buffer containingan inhibitor cocktail (PKC inhibitor peptide, PKA inhibitor peptide, andCompound R24571) to block possible contaminating non-ERK kinases. Whereappropriate, varying concentrations (0.1, 1, and 10 μM) of CDDO orCDDO-Me was added. Dephosphorylated myelin basic protein (MBP; 25 μg)was used as substrate. Phosphorylation of MBP was observed by using ananti-phospho-MBP antibody. As a negative control, a lysate containinginactive ERK (obtained from K562 cells treated for 4 hrs in vivo with 10μM of MEK inhibitor PD58059) was used in the assay. The amount of ERK2immunoprecipitated from each sample was determined by using anti-ERK2antibody.

[0301] For K562 cells, a control was performed to determine that CDDO orCDDO-Me could at least inhibit ERK upstream, if not directly. K562 cellswere treated in vivo for 4 hrs with 1 μM CDDO-Me and lysate from thesecells was used in the in vitro kinase assay.

[0302] Immunophenotyping. The PE-conjugated anti-CD11b, FITC-conjugatedanti-CD14 monoclonal antibody (mAb) (Becton Dickinson) and PE-conjugatedanti-CD95 mAb (PharMingen) were used at a {fraction (1/10)} dilution.The percentage of positive cells was calculated by subtracting thepercentage of cells with a fluorescence intensity greater than the setmarker using the isotype control (background) from the percentage ofcells with a fluorescence intensity greater than the same marker usingthe specific antibody.

[0303] Annexin V staining. Cells were washed in phosphate-bufferedsaline (PBS) and resuspended in 100 μl of binding buffer containingAnnexin V (Roche Diagnostic Corporation, Indianapolis, Ind.). Cells wereanalyzed by flow cytometry after the addition of propidium iodide (PI).Annexin V binds to those cells that express phosphatidylserine on theouter layer of the cell membrane, and PI stains the cellular DNA ofthose cells with a compromised cell membrane. This allows for live cells(unstained with either fluorochrome) to be discriminated from apoptoticcells (stained only with annexin V) and necrotic cells (stained withboth annexin and PI).

[0304] Cytofluorometric analysis of the ΔΨ_(m). To evaluate the ΔΨ_(m),cells were incubated with the cationic lipophilic dyechlorophenyl-X-rosamine (CMXRos; 150 nM; Molecular Probes, Inc., Eugene,Oreg.). CMXRos is incorporated into mitochondria driven by the ΔΨ_(m)and reacts with thiol residues to form covalent thiol ester bonds.CMXRos fluorescence was recorded by flow cytometry in the FL3 channel.Background values of the apoptosis of control cells cultured without theCDDO-Me or in DMSO-solvent control (<10% CMXRos-low) were subtractedfrom the values obtained under the experimental conditions.

[0305] In a series of experiments, cells were pretreated for 1 hour with50 μM CyA or 50 μM BA prior to the addition of CDDO-Me. These agentsprevent a reduction in ΔΨ_(m) induced by various agents that openmitochondrial permeability transition pores.

[0306] Detection of active caspases. The cell-permeable fluorogenicsubstrate Phi-Phi-Lux-G1D2 was administered to monitor caspase activityaccording to the manufacturer's recommendations (Oncolmmunin, Inc,Kensington, Md.). Briefly, 10⁶ cells were resuspended in 5 μL ofsubstrate solution and incubated for 1 hour at 37° C. in the dark. Afterincubation, cells were washed, and the fluorescence emission wasdetermined using the FL-1 channel of a Becton Dickinson FACScan flowcytometer.

[0307] Statistics. Results are expressed as means±SEM. Levels ofsignificance were evaluated by a two-tailed paired Student's t test, andP<0.05 was considered significant.

Example 2 Anticancer Properties of CDDO-Compounds and CombinationTherapies Thereof

[0308] Two CDDO-compounds, 2-Cyano-3,12-Dioxoolean-1,9-Dien-28-Oic Acid(CDDO) and its C-28 methyl ester, (CDDO-Me) induce differentiation,inhibit cell growth and induce apoptosis in leukemia cell lines and inprimary samples from patients with AML.

[0309] Described herein are the mechanisms and efficacy of theseCDDO-compounds activities in AML which allows the use of theseCDDO-compounds as drugs for the treatment of hematological malignancies.Growth-inhibitory effects of CDDO and CDDO-Me on primary AML inclonogenic assay systems including the NOD/Scid model of AML aredescribed. The contributions of the mitochondrial (Bcl-2-regulated) anddeath receptor (Fas/Fas-L) pathways for the induction of apoptosis byCDDO and CDDO-Me are described. A decrease in Bcl-2 expression isdemonstrated.

[0310] The inventors also show that CDDO and CDDO-Me are novel ligandsfor PPARγ which is highly expressed in AML. Also demonstrated is asynergism between CDDO-compounds and retinoids. As PPARγ formsfunctional heterodimers with RXR, optimal combination of CDDO-compoundsand retinoids for induction of apoptosis in AML are provided. Finally,pharmacokinetic, tissue distribution and toxicity studies ofCDDO-compounds in mice for preclinical development of this combinationtherapy are described.

[0311] CDDO-Compounds Decrease Viability and Induce Apoptosis inLeukemic Cell Lines:

[0312] CDDO. The effect of CDDO on the survival of HL-60 and U937leukemia cell lines is depicted in FIG. 1. A time-and dose-dependentdecrease of HL-60 cells: at 48 hours, 10⁻⁸ M, at 72 and 96 hours, 10⁻⁹ Mof CDDO reduced cell numbers by 50%. Viability of U937 cells wasinhibited at 10⁻⁷ to 10⁻⁸ at 48 hours. DNA fragmentation was measured toconfirm that the cell death induced by CDDO was due to apoptosis on asub G₁ cell population by DNA/RNA flow cytometry (FIG. 2). Thepercentage of proliferating cells were analyzed by FCM. A significantdecrease was noted at 10⁻⁷ M, as compared to DMSO at 48, 72 and 96 hoursin HL-60 cells. In U937 cells, the proliferating (S+G₂M) fractiondecreased at 10⁻⁷ M (14.9% compared with 23.3% in DMSO-controls) and at10⁻⁶ M (5.6%) (FIG. 2).

[0313] As HL-60 cells do not have functional p53 (p53-‘null’) thecytotoxic effect of CDDO is p53-independent. Also, HL-60-Dox cells withhigh expression of the MDR-1 gene were sensitive to CDDO-inducedkilling, and blocking MDR-1 by the specific inhibitor PSC-833 did notaffect CDDO cytotoxicity. Hence, MDR-1 does not seem to affect CDDOkilling.

[0314] The effect of CDDO on the viability of a variety of otherleukemia cell lines is shown in FIG. 3. Exposure to 10⁻⁸ M to 10⁻⁵ MCDDO induced apoptosis in HL-60-Dox and TF-1 cells. CDDO treatment alsoenhanced ara-C-induced killing of leukemic cells as shown in HL-60-DOXcells (FIG. 4).

[0315] CDDO induces of differentiation in U937 leukemic cells (Suh etal., 1999). Thus, induction of monocytic differentiation in HL-60 cellswas determined morphologically and by the induction of CD14 expression(see FIG. 16).

[0316] CDDO induced apoptosis without inducing differentiation inseveral leukemic cell lines and in primary AML, however CDDO inducedapoptosis in HL-60 cells following induction of differentiation. Thus,CDDO can affect both apoptosis and differentiation in different cellulartargets.

[0317] CDDO-Me. CDDO-Me is consistently more active than CDDO, with IC₅₀of 0.4, 0.4 and 0.27 μM in the leukemic cell lines HL-60, KG-1 and NB4.Profound cytotoxic effect in Daudi lymphoid leukemic cells, were seenand 0.3 μM CDDO-Me decreased the cell number to 22.5% of DMSO-controlsat 48 hrs. Mechanism of growth inhibition was analyzed on cell cycle andapoptosis in HL-60 cells. As demonstrated in FIG. 20A, CDDO-Me inhibitedcell growth at 0.05 and 0.1 μM in a dose- and time-dependent fashion. At0.5 μM essentially no viable cells were recovered at 48 hrs. Cell cyclemeasurements revealed no significant differences in cell cycledistribution. To study the effect of CDDO-Me on apoptosis, HL-60 cellswere stained with FITC-labeled annexin V (Vermes et al., 1995). Cellswere simultaneously stained with PI and analyzed by flow cytometry: adose-dependent increase in annexin V binding in CDDO-Me-treated cells(FIG. 20B) were seen. Thus, induction of apoptosis is the primarymechanism of CDDO-Me-induced growth arrest. Again as with CDDO, thecytotoxic effect of CDDO-Me is p53-independent. Also, HL-60-Dox cellswith high expression of the MDR-1 gene were sensitive to CDDO-Me-inducedkilling, and blocking MDR-1 by the specific inhibitor PSC-833 did notaffect CDDO-Me cytotoxicity. Hence, CDDO-Me is p53 and MDR-1independent.

[0318] CDDO-Me is a more potent inducer of granulo-monocyticdifferentiation in HL-60 cells as compared with CDDO: at 0.1 μM ofCDDO-Me 86.6% of cells were CD11b+, while 1 μM of CDDO is needed toexert similar effect. In different leukemic cell lines and in primaryAML, CDDO-Me induced apoptosis without marked differentiation, whereasin HL-60 cells apoptosis was observed primarily in differentiated cells.Thus, CDDO-Me affects both apoptosis and differentiation in differentleukemic cell populations.

[0319] CDDO-Compounds Decrease Viability and Induce Apoptosis in PrimaryAML Cells in Suspension Cultures and in Clonogenic Assays:

[0320] CDDO. In primary AML, CDDO and CDDO-Me induced apoptotic celldeath in a dose-dependent fashion as determined by subG₁ flow cytometry.1 μM CDDO induced apoptosis in 3 of 6 AML samples in vitro, and 5 μMinduced apoptosis in 5 of 6 samples (FIG. 5). CDDO increasedara-C-induced apoptosis in AML samples (DMSO control, 27.7±3.7%; CDDO 1μM, 41.2±4.4%; ara-C 1 μM 41.6±4.6%; CDDO+ara-C, 53±4.6%, n=23). Thepaired mean difference±SEM was 13.5%+2.8%. Monocytic differentiation wasinduced in {fraction (3/11)} AML, as evidenced by downregulation of CD34expression and induction of the monocytic differentiation marker CD14.This effect was more pronounced in ara-C/CDDO combinations (FIG. 6).

[0321] The effect of CDDO on clonogenic AML cells were tested. Colonyformation of AML progenitors (n=6) was significantly reduced at 1 μMCDDO (58±4.6%). 0.5 μM CDDO resulted in inhibition of >50% colonies inone CML-BC sample tested. MACS-separated normal CD34⁺ cells were used ascontrols, CFU-GM of normal CD34⁺ cells were inhibited only by 26.8±4.3%at this concentration (n=3) [p=0.004] (FIG. 7).

[0322] Thus, CDDO decreases viability and induces apoptosis in myeloidleukemic cell lines and primary AML samples. CDDO also inducesdifferentiation in HL-60 cells and in primary AML. CDDO also reducescolony formation of AML progenitors, but did not inhibit normal CFU-GMcells.

[0323] CDDO-Me. In primary AML, CDDO-Me induced apoptotic cell death ina dose-dependent fashion as determined by DNA flow cytometry (subG₁): at1 μM CDDO-Me, apoptosis was induced in 5 of 6 AML samples in vitro, and5 μM induced apoptosis in 6 of 6 samples. More than 90% apoptotic cellswere detected in {fraction (4/6)} samples following exposure to 5 μM ofCDDO-Me (FIG. 21A). At lower concentrations, CDDO-Me induced adose-dependent increase in the percentage of the apoptotic cells in{fraction (4/4)} samples tested (FIG. 21B). The paired mean differencebetween the CDDO-Me 0.1 μM and DMSO control is 26.9%±10.8%(CDDO-Me-DMSO, mean±SEM), and the paired mean difference between theCDDO-Me 0.5 μM and DMSO is 43.2%±5.2%. CDDO-Me also enhancedara-C-induced cell killing (DMSO control, 24.9±7.4% CDDO-Me 1 μM,50.5±15%; ara-C 1 μM 39.8±8.2%; CDDO-Me±ara-C, 65.4±10.2%, n=6).Monocytic differentiation was induced in {fraction (2/5)} AML, asdemonstrated by induction of the monocytic differentiation marker CD14.CDDO-Me also induced apoptosis in CML-blast crisis samples in vitro (in3 of 4 samples at 1 μM, in 4 of 4 at 5 μM), enhanced ara-C-induced celldeath and induced differentiation in 1 of 4 samples tested. Effects ofCDDO-Me were also tested on clonogenic AML cells. Colony formation ofAML progenitors was significantly inhibited in a dose-dependent fashion,with 46.7%±6.6% surviving colonies at 0.1 μM of CDDO-Me, 27.5%±6.8% forthe CDDO-Me 0.3 μM, and 8.8%±3.8% for the CDDO-Me 0.5 μM (FIG. 22). 0.1μM CDDO-Me also inhibited >50% colonies in one CML-BC sample tested. Incontrast, 79±5.6% and 63.4±1.1% CFU-GM generated from normal CD34* cellswere detected after 0.1 and 0.5 μM of CDDO-Me treatment (n=3).

[0324] Thus, CDDO-Me decreases viability and induces apoptosis inmyeloid leukemic cell lines and in primary AML samples. CDDO-Me alsoinduces differentiation in HL-60 cells and in primary AML. CDDO-Me alsosignificantly reduced colony formation of AML progenitors.

[0325] CDDO-Compounds Induce Changes in the Apoptotic Machinery:

[0326] The molecular mechanisms underlying CDDO-compound induced celldeath were observed by staining HL-60-Dox cells with annexin V, whichbinds phosphatidylserine with high affinity. Translocation ofphosphatidylserine to the cell surface is considered one of the earliestevents in apoptosis and can be analyzed by staining with FITC-labeledannexin V (Vermes et al., 1995). Cells were simultaneously stained withPI and analyzed by flow cytometry.

[0327] CDDO. A time-dependent increase in annexin V binding in CDDOtreated cells at 24 and 48 hours was observed (FIG. 8A). To test theeffect of CDDO on the cleavage of caspase-3 the fluorogenic substrate ofcaspase-3 Phi-Phi-Lux (PI) was used. Cells were simultaneously stainedwith PI and analyzed by flow cytometry. As shown in FIG. 8A,CDDO-compounds induced Phi-Phi-Lux-positivity that paralleled changes inthe plasma membrane. Similar results were obtained in U937 cells, whereincreases in annexin-V and Phi-Phi-Lux positivity were seen 48 hrs aftertreatment with 1 μM CDDO. Early (24 hrs) changes in mitochondrialmembrane potential preceded caspase activation and peaked at 48 hrs(FIG. 8B).

[0328] To study the molecular mechanisms underlying CDDO-induced celldeath, time-dependent apoptotic changes in D9 leukemic cells wereanalyzed. Translocation of phosphatidylserine to the cell surface isconsidered an early event in apoptosis and can be analyzed by stainingwith FITC-labeled annexin V. A time-dependent increase in annexin Vbinding in CDDO-treated cells at 5 and 24 hours was observed. To testthe effect of CDDO on the cleavage of caspase-3, the fluorogenicinhibitor that binds to activated caspase-3 (Caspatag, Intergen), wasutilized. CDDO induced Caspatag positivity that paralleled changes inthe plasma membrane. Relatively early (3 and 5 hrs) changes inmitochondrial membrane potential were observed (measured by CMXRos).These data demonstrate that CDDO induces apoptotic cell death throughthe mitochondrial pathway.

[0329] The effect of CDDO on the processing of different caspases wasalso analyzed. In AML-2 cells, CDDO induced a dose-response decrease inpro-caspase-3, with appearance of cleaved caspase-3 product at 1 μM ofCDDO at 24 hrs. CDDO (2, 5 and 10 μM) also induced decrease inunprocessed caspase-8 and -9. This activation of caspases correlatedwith a marked decrease in cell viability.

[0330] To characterize the correlation between PPARγ and caspaseactivation, caspase-3 levels in D9 cells transfected with wt-PPARγ wereanalyzed. Six different single cell-derived clones were treated with 3μM CDDO or vehicle controls for 5 hrs. Caspase-3 cleavage was analyzedby Western blot. A decrease in pro-caspase-3 with appearance of thecleaved product was readily observed in all clones. The degree ofcleavage was higher in PPARγ-transfected than in vector control cellsthat express endogenous PPARγ. Analysis of DNA fragmentation revealedendonucleolytic DNA cleavage in CDDO-treated cells that waspredominantly seen in PPARγ-transfected cells. These changes correlatedwith plasma membrane changes: vector control=47.0% annexin V-positivecells; wt-PPARγ-transfected cells=73.3±1.1% annexin V (+), n=6. Cellnumber did not change in vector control cells, while it decreased by48.8±12.4% in CDDO-treated PPARγ-transfectants. PPARγ-transfected cellswere also more sensitive to PPARγ-ligand 15d-PGJ2 (41.0% vs 16.0%annexin (+) cells at 3 μM). These results demonstrate that caspaseactivation by CDDO depends on the expression levels of PPARγ.

[0331] In addition, a time-dependent increase in annexin V binding inCDDO treated cells at 24 and 48 hours was observed (FIG. 8A). To testthe effect of CDDO on the cleavage of caspase-3 the fluorogenicsubstrate of caspase-3 Phi-Phi-Lux (PI) was used. Cells weresimultaneously stained with PI and analyzed by flow cytometry. As shownin FIG. 8A, CDDO-compounds induced Phi-Phi-Lux-positivity thatparalleled changes in the plasma membrane. Similar results were obtainedin U937 cells, where increases in annexin-V and Phi-Phi-Lux positivitywere seen 48 hrs after treatment with 1 μM CDDO. Early (24 hrs) changesin mitochondrial membrane potential preceded caspase activation andpeaked at 48 hrs (FIG. 8B).

[0332] Mechanisms of CDDO-induced apoptosis in leukemia cell lines andin primary AML samples were also determined. The involvement of thedeath receptor/Fas pathway was studied in NB4 cells. Treatment with 0.5μM CDDO induced cell surface Fas/CD95 expression at 48 hours (MFI 34.8vs 24.8 in DMSO-treated cells and 26 in untreated control). At thisconcentration, 22.3% of cells were annexin V positive compared with11.4% in DMSO-controls. At 1 μM, 56.3% of cells were apoptotic.Similarly, 1 μM CDDO induced Fas expression in {fraction (7/12)} AMLCD34+ cells (MFI, 22.3±3.5 in DMSO-controls, 40±4.3 in CDDO-treatedCD34+ cells, p<0.01). However, CDDO did not induce Fas expression inp53-negative HL-60 and HL-60-Dox cells (Owen-Schaub et al., 1994),suggesting involvement of the different mechanisms of the apoptotic celldeath in these cells.

[0333] The involvement of the mitochondrial apoptotic pathway in CDDOmediated apoptosis was analyzed in U937 cells. A >40% decrease in Bcl-2protein was observed at 72 hrs of 1 μM of CDDO treatment as determinedby quantitative flow cytometry. Decreased Bcl-2 expression was alsodemonstrated by Western blot analysis in CDDO-treated HL-60, KG-1 andNB4 cells. CDDO also significantly decreased Bcl-2 expression level inCD34+ cells in 9 of 18 primary AML samples tested (p=0.01).

[0334] The effect of CDDO on the Bcl-2 mRNA expression was analyzedutilizing quantitative TaqMan PCR. In HL-60 cells, Bcl-2 mRNA decreasedat 24 hrs and decreased further (>than 10-fold) at 72 hours; in NB4cells, a small decrease of Bcl-2 mRNA (2-fold) was observed only at 72hrs (FIG. 9). These results were confirmed in two independentexperiments.

[0335] The expression of the other anti-and pro-apoptotic proteinsfollowing CDDO exposure were also analyzed. No differences were observedin the expression of the pro-apoptotic proteins Bax and Bcl-Xs. However,the protein levels of the inhibitor-of-apoptotic protein XIAP decreasedin KG-1 and NB-4 cells. A decrease in XIAP mRNA levels at 48 and 72 hrswas also demonstrated (FIG. 10).

[0336] The inventors also tested if overexpression of the anti-apoptoticprotein Bcl-2 protected leukemic cells from CDDO-induced cytotoxicity.For these experiments U937 cells transduced with Bcl-2 were used withempty-vector containing counterparts as controls. Bcl-2 overexpression(3-fold) did not protect cells from CDDO-killing.

[0337] The differential expression of a large number of genes involvedin apoptosis, proliferation and signal transduction were analyzedutilizing the Atlas Cancer c-DNA array from Clontech (Palo Alto,Calif.). Leukemic U937 cells were treated with CDDO for 24 hrs (1 μM) tostudy changes in gene expression levels prior to the induction ofdifferentiation and/or apoptosis. The downregulation of 17 genes (overtwo-fold) was seen, some of these genes were MAPKK5, STAT5A and B, DNAtopoisomerase 2, VEGFR1 and VEGF. 7 genes were upregulated includingJNK2 and death receptor DR5, a receptor for TRAIL.

[0338] Thus, it is demonstrated that CDDO downregulates anti-apoptoticBcl-2 and XIAP mRNA and protein expression level and induces Fasexpression in leukemic cell lines and in the majority of primary AMLsamples. The cDNA array data provides a means for the identification ofgenes contributing to the anti-leukemic effects of CDDO.

[0339] Differential expression of a large number of genes involved inapoptosis, proliferation and signal transduction was also performed forCDDO-Me. Leukemic U937 cells were treated with 1 μM of CDDO-Me for 1hour with the aim of inducing changes in gene expression levels prior tothe induction of differentiation or apoptosis. In preliminary studies,the inventors observed over two-fold downregulation of 23 genes: theseinclude MAPK/ERK2, endothelin2, MLH1. Remarkably, decreased expressionof VEGFR1, that potentially mediate pro-angiogenic properties of theleukemic cells, was observed (FIG. 33). Twenty-two genes wereupregulated including c-jun, TNF-R1 and CPBP, zinc finger proteininvolved in transcriptional control. The cDNA array data requireconformation but may indicate what changes in gene expression contributeto the observed anti-leukemic effects of CDDO-Me.

[0340] PPARγ, a member of a family of nuclear receptors, inducesdifferentiation and growth arrest in preadipocyte cells and can mediateinflammatory processes. The PPARγ ligands 15d-PGJ2 at 3 μM and BRL49653at 5 μM induce monocytic differentiation of HL-60 cells but exerted nokilling at this concentration (Tontonoz et al., 1998). The ability ofCDDO-compounds to directly interact with PPARγ was assessed by ascintillation proximity assay (SPA) (Nichols et al., 1998) using³H-rosiglitazone as the ligand and bacterially expressed PPARγ LBD. Asshown in FIG. 11A, non-radioactive CDDO competed efficiently with[³H]-rosiglitazone for binding to the PPARγ LBD (K_(i)=12 nM) (Wang etal., 2000). In a similar assay, rosiglitazone was shown to compete forbound ³H-CDDO, with K_(i) values of 50 nM.

[0341] To determine if bound CDDO transactivates PPARγ, a Gal4-PPARγchimeric protein was used to drive the expression of luciferase linkedto the DNA binding sequence of GAL4 (FIG. 11B) (Wang et al., 2000). CDDOtransactivates GAL4-PPARγ in a dose-dependent manner. In contrast, CDDOdid not transactivate the PPARα receptor. These data demonstrate thatCDDO can interact directly and specifically with PPARγ LBD atsub-micromolar concentrations and thus establishes CDDO as a novel PPARγligand. All known pharmacological PPARγ ligands had anti-proliferativeand differentiating effects only when used at higher (3 or 5 μM)concentrations.

[0342] Western Blot Analysis. The expression of PPARγ in leukemic celllines and primary AML/MDS samples was analyzed by western blot. Thespecificity was confirmed using a monoclonal antibody to PPARγ (SantaCruz Biotechnology, Inc.) or an antibody pre-adsorbed with a blockingpeptide for competition studies. The monoclonal antibody reacted withboth, PPARγ1 and PPARγ2 isoforms. Disappearance of the specific bandafter pre-adsorbtion with blocking peptide confirmed the correctposition of the PPARγ band (FIG. 12A). The PPARγ protein was expressedin 6 myeloid and 6 lymphoid cell lines tested (FIG. 12B, FIG. 13B).PPARγ was also detected in {fraction (9/11)} primary AML samples withhigh (>50%) blast count, low expression was noted in 2 of 4 samples frompatients with advanced MDS (RAEB) (FIG. 13). PPARγ was not expressed in2 samples of normal magnetic-separated CD34⁺ cells (FIG. 13). Thus,PPARγ is highly expressed in leukemic blasts.

[0343] Sensitivity to CDDO-induced apoptosis correlated with levels ofPPARγ: in U937 cells expressing 3-fold higher PPARγ levels than HL-60cells, 1 μM CDDO induced 69.4% apoptotic sub-G₁ cells at 48 hrs comparedwith 34.8% in HL-60. In contrast, 73.6% Daudi cells which have lowestdetectable PPARγ expression were alive at this concentration of CDDO.However, at higher (2 μM) CDDO concentrations both cell lines werekilled equally (30.2 and 25.3% viable cells) perhaps as the result ofmaximal receptor stimulation.

[0344] To test if the cytotoxic effect of CDDO is mediated by PPARγreceptor, growth of Daudi cells (which have lowest detectable PPARγexpression) following CDDO treatment was examined. In U937 cells only44.7% of the cells remained viable after 48 hrs of CDDO (1 μM)treatment. In contrast, 73.6% Daudi cells were alive at thisconcentration of CDDO. However, at higher (2 μM) CDDO concentrationsboth cell lines were killed equally (30.2 and 25.3% viable cells)perhaps as the result of maximal receptor stimulation.

[0345] To characterize the correlation between PPARγ and caspaseactivation, caspase-3 levels in D9 cells transfected with wt-PPARγ wereanalyzed. Six different single cell-derived clones were treated with 3μM CDDO or vehicle controls for 5 hrs. Caspase-3 cleavage was analyzedby Western blot. A decrease in pro-caspase-3 with appearance of thecleaved product was readily observed in all clones. The degree ofcleavage was higher in PPARγ-transfected than in vector control cellsthat express endogenous PPARγ. Analysis of DNA fragmentation revealedendonucleolytic DNA cleavage in CDDO-treated cells that waspredominantly seen in PPARγ-transfected cells. These changes correlatedwith plasma membrane changes: vector control=47.0% annexin V-positivecells; wt-PPARγ-transfected cells=73.3±1.1% annexin V (+), n=6. Cellnumber did not change in vector control cells, while it decreased by48.8±12.4% in CDDO-treated PPARγ-transfectants. PPARγ-transfected cellswere also more sensitive to PPARγ-ligand 15d-PGJ2 (41.0% vs 16.0%annexin (+) cells at 3 μM). These results demonstrate that caspaseactivation by CDDO depends on the expression levels of PPARγ.

[0346] It has recently been demonstrated that PPARγ ligands recruit theDRIP205 coactivator to PPARγ (Yang et al., 2000). DRIP205 is a keysubunit of the DRIP multisubunit coactivator complex that anchores theother 14 subunits to the nuclear receptor LBD. DRIP205 was identifiedfrom U937 leukemic cells (Rachez et al., 2000). DRIP205 expression wasfound to be low in parental D9 and in vector-transfected cells, while itwas higher in selected sub-clones transfected with wt-PPARγ and wasfurther indu ced by PPARγ ligand 15d-PGJ2.

[0347] To test the efficacy of CDDO compared to other PPARγ ligands, theeffect of PPARγ ligands such as 15d-PGJ2 (Cayman Chemical Company),BRL49653 (Smith Kline Beecham), L-805645 (Merck), GW347845X (GlaxoWelcome) were compared to that of CDDO: 5 μM of 15d-PGJ2 was requiredfor 50% inhibition of HL-60 cells; other ligands including roziglitazone(BRL49653) exerted similar effects only at high (25-50 μM)concentrations. This decrease in cell viability was mediated byinduction of apoptosis, as determined by annexin V staining. Ofimportance, RXR-specific ligand LG100268 enhanced growth-inhibitoryeffects of PPARγ ligands.

[0348] In HL-60 cells, all PPARγ ligands induced CD11b expression in adose-dependent fashion, but significantly higher concentrations wererequired to achieve effects similar to CDDO (Table 4): TABLE 4Expression of CD11b in HL-60 cells treated with PPAR_(γ) ligands for 7days: 15d- Control CDDO PGJ2 GW347845X L-805645 BRL49653 (DMSO) μM CD11b% M CD11b % μM CD11b % μM CD11b % μM CD11b % 0.1 45.0 1 34.1 10 28.3 1038.6 10 38.9 24.9 0.3 75.9 3 49.4 25 44.8 25 42.9 25 48.5 0.5 83.1 583.9 10 88.9

[0349] In primary AML samples, exposure to PPARγ ligands 15d-PGJ2 (5μM), BRL49653 (25 μM), L-805645 (25 μM), GW347845X (25 μM) induced27.7%, 7%, 21% and 27.8% annexin-V-positive cells, respectively at 72hrs; this effect was enhanced by combination with the RXR-specificligand LG-10268. These data demonstrate the ability of PPARγ ligands toinduce apoptosis and differentiation and also provide evidence that CDDOhas higher activity in leukemic cells than all other PPARγ ligands.

[0350] Genetic Analysis. To further characterize the correlation betweenhPPARγ and antileukemic activity, HL-60, CDM-1, U937, KG-1 and KBM-3cells were transfected with an empty expression vector (pcDNA3),FLAG-tagged wt-PPARγ or FLAG-tagged L466A/E469A dominant-negative (DN)PPARγ mutant together with a selectable marker (neo). In the DN-PPARγmutant highly conserved hydrophobic and charged residues (Leu⁴⁶⁶ andGlu⁴⁶⁹) in helix 12 of the ligand-binding domain were mutated toalanine. It retains ligand and DNA binding, but exhibits reducedtransactivation due to impaired coactivator (CBP and SRC-1) recruitment.

[0351] wt-hPPARγ and the DN-PPARγ mutant constructs were sequenced priorto transfection to verify the correct sequence. Plasmid DNA was purifiedusing the QIAprep spin miniprep kit (Qiagen). Stable transfection of theleukemic cells was performed using the calcium-phosphate method. Cellswere split the day before transfection. On the day of transfection,7.5×10⁶ cells were harvested by centrifugation and seeded in 5 ml ofgrowth medium supplemented with serum and antibiotics. 0.5 ml ofcalcium-phosphate-DNA (5 μg) precipitate mixture was added to cells andincubated overnight. The next day medium containing complexes wasremoved, cells were washed in PBS and resuspended in 5 ml of freshmedium. G418 was added at 48 hrs post-transfection; G418 concentrationswere selected for each cell line based on the preliminary dose-responsecurve. After 4 days cells were passaged at 1:5 into the G418-containingselective medium. Medium was replaced on day 3-4, and cells were platedat 100 μl/well in a 96-well plate. The cultures were re-fed every 3-4days with addition of fresh medium containing G418. When culturesreached 50% confluence, cells growing in the individual wells (clones)were collected, transferred into a 24-well plate and screened for theexpression of the transgene utilizing 2 methods: 1) Dot-blot analysis ofprotein by direct deposition of the sample (protein lysate) on themembrane followed by western blot with anti-PPARγ antibody. 2) Westernblot analysis of the clones selected by dot-blot analysis, usinganti-PPARγ and anti-FLAG antibodies.

[0352] Twenty clones were tested for pcDNA3, wt- and DN transfectants;the 2 best clones were selected for further cloning. To obtainsingle-cell clones, cells were diluted to 0.8 cells/well (i.e., plating100 μl/well of 8 cells/ml dilution) and plated in a 96-well plate; thisdilution provides 36% of wells with 1 cell/well by Poisson statistics.As controls, 10 wells were plated with 100 μl/well of 80 cells/mldilution (i.e., 8 cells/well).

[0353] After sub-cloning the analysis of protein expression was repeatedfor the selected subclones (dot-blot followed by Western blot analysis),and the presence of transgene was confirmed by RT-PCR for neomycin(neomycin ORF: bases 2151-2945 in pcDNA3). Oligonucleotide primers (F,forward; R, reverse) used were as follows: F 5′-CAAGATGGATTGCACGCAGG-3′and R 5′-GAGCAAGGTGAGATGACAGG-3′. Amplified products (325 bp) wereseparated by gel electrophoresis.

[0354] HL-60, CDM-1, KG-1, KBM-3 and D9 cells were transfected with wt-,DN-PPARγ and vector control (pcDNA3). CDM-1 and KG-1 cells do notexpress endogenous PPARγ, while HL-60, D9 and KBM-3 express variablelevels of the protein. For HL-60 cells, {fraction (120/176)} wells werepositive for wt-PPARγ, {fraction (141/178)} for DN-PPARγ and {fraction(202/384)} for pcDNA; for CDM-1 cells, {fraction (130/171)} wells werepositive for wt-PPARγ, {fraction (132/178)} for DN-PPARγ and {fraction(123/180)} for pcDNA. 20 clones each (wt-, DN-PPARγ and pcDNA) weretested by dot-blot and verified by Western blot analysis. Two selectedclones/each were further subcloned; analysis of subclones is currentlyin progress using dot-blot, Western blot and RT-PCR.

[0355] The sensitivity of leukemic cells that overexpress PPARγ toCDDO-induced killing was tested. Vector control (pcDNA) orwt-PPARγ-transfected D9 cells (2 different clones) were treated 0.2 μMof CDDO for 48 hrs. Cell viability was assessed by cell count afterTrypan blue exclusion, and apoptosis determined by PS/annexinV/Propidium Iodide (PI) flow cytometry. Leukemic cells transfected withPPARγ expressed 2.8-3.7-fold higher amounts of protein compared withvector controls. As expected, forced expression of PPARγ significantlyenhanced the sensitivity of leukemic cells to CDDO killing. Highexpression of PPARγ in transfected cells was confirmed byimmunohistochemistry analysis demonstrating high levels of nuclear PPARγin both transfected clones. Importantly, CDDO increased PPARγ expressionin both, vector control and transfected cells. This experiment providesadditional evidence for CDDO being an effective PPARγ ligand and alsoprovides an explanation for activity of the compound in cells with lowbaseline PPARγ levels. No significant differences in Bcl-2 and Baxexpression were noted in PPARγ- or vector-transfected cells. These dataalso indicate that PPARγ expression determines the sensitivity ofleukemic cells to CDDO-induced apoptosis. Of note, similar results (>50%higher sensitivity compared to vector-control cells) were observed inall six single cell-derived subclones tested.

[0356] CDDO-Me. The annexin/PI fluorimetric assay demonstrated atime-dependent increase in annexin V binding in CDDO-Me-treated cells(FIG. 23). Caspase-3 has been shown to play a pivotal role in theexecution of programmed cell death induced by different stimuli (Ibradoet al., 1996; Ohta et al., 1997; Schlegel et al., 1996). Effects ofCDDO-Me on the cleavage of caspase-3 were analyzed utilizing thefluorogenic substrate of caspase-3 Phi-Phi-Lux. As demonstrated in FIG.24A, treatment of U937 cells with 1 μM CDDO-Me for 6 hours resulted in67% of Phi-Phi-Lux positive/PI negative cells, indicating caspasecleavage. Activation of caspase-3 resulted in the appearance of the17-kD proteolytic product of caspase-3 and complete disappearance ofuncleaved 32-kD caspase-3 after 6 hours by Western blot analysis.Importantly, pre-treatment of U937 and HL-60 cells with 25 μM ofcaspase-3 inhibitor Z-DEVD-fmk for 1 hour significantly reduced annexinV-positivity (FIG. 25) and specifically diminished cleavage ofPhi-Phi-Lux (from 15.1% to 2.7%). Western blot analysis using specificantibody for cleaved caspase-3 also demonstrated disappearance of theband (FIG. 25). These data confirm the key role of caspase-3 activationin the CDDO-Me-induced apoptosis.

[0357] An early mitochondrial disruption has been observed in a numberof different models of apoptosis. Changes in the cellular content of thecationic lipophilic fluorochrome CMXRos were determined by FACS analysisto measure loss of the mitochondrial membrane potential (ΔΨ) (Macho etal., 1996). U937 cells exhibited a time-dependent decrease in ΔΨ (FIG.24B). Similarly, exposure of HL-60 to CDDO-Me (1 μM) for 2 and 4 hrsincreased the number of cells with low JC-1 (57 and 60%) or CMXRosstaining (46 and 57%), both assays measuring the decrease in ΔΨ.CDDO-Me-induced changes in the mitochondrial membrane potential of thecells preceded caspase activation and changes in the composition of theplasma membrane. In order to validate the role of the dissipation of ΔΨin CDDO-treated cells the pharmacological inhibitors of permeabilitytransition cyclosporin A (CyA) (Nicolli et al., 1996) (10μM, Sandoz) andbongkrekic acid (BA) (Marchetti et al., 1996) (Calbiochem, CA) wereused. Addition of CyA partially inhibited CDDO-Me-triggered ΔΨ loss,providing further evidence for effects of CDDO-Me on ΔΨ (FIG. 26). Thesame effect was observed using BA (52% in CDDO-Me treated HL-60 cells,23% when cells were pre-treated with BA). Notably, pretreatment withcaspase inhibitor Z-DEVD-fmk also prevented appearance of cells withdecreased ΔΨ (40% in CDDO-Me-treated cells compared with 4% after DEVDpretreatment) in U937 cells suggesting the existence ofcaspase/mitochondria amplification loop. Collectively, these datademonstrate that CDDO-Me induces apoptotic cell death through activationof caspases and effects on the cellular mitochondrial potential.

[0358] The inventors then examined if CDDO-Me induced cell death bymodulating the mitochondrial or the death receptor pathways ofapoptosis. Thus, effect of CDDO-Me on Bcl-2 protein expression werestudies. Prolonged (5 days) treatment with low concentrations of CDDO-Me(0.05 and 0.075 μM) did not significantly affect Bcl-2 expression levelsin HL-60 and NB4 cells despite substantial cell killing, pointing toalternative mechanisms of induction of apoptosis by CDDO-Me (FIG. 27A).Bax functions as a promoter of cell death and its upregulation has beenassociated with enhanced apoptosis (Bargou et al., 1995; Yin et al.,1997). Treatment of HL-60 cells with CDDO-Me resulted in increasedlevels of Bax protein starting at 2 hrs with simultaneous cleavage ofcaspase-3 as determined by Western blot analyses (FIG. 27B). Bax levelswere also induced when HL-60 and NB4 cells were treated for 5 days asdescribed above (FIG. 27A). Bcl-2 and Bax are known to form heterodimers(Sato et al., 1994), and the ratio of anti-apoptotic versus proapoptoticdimers or the amount of free Bax are thought to be important indetermining resistance of cells to apoptosis. In immunoprecipitationstudies the inventors observed increased levels of Bcl-2 bound to Baxafter 24 hrs of CDDO-Me indicating elimination of Bcl-2 anti-apoptoticfunction through binding to Bax. Effects of CDDO-Me on mRNA Bax levelswere tested. As determined by Northern blot analysis, CDDO-Me treatmentinduced Bax mRNA in both HL-60 and U937 cells (FIG. 27C), hence CDDO-Memay affect transcriptional regulation of Bax.

[0359] To study if the overexpression of Bcl-2 can protect leukemiccells from CDDO-Me-induced cytotoxicity the inventors used U937 cellstransduced with Bcl-2 (Vrana et al., 1999) and their empty-vectorcontaining counterparts. Bcl-2 overexpression (3-fold) did not protectcells from CDDO-Me-killing. However, HL-60 cells overexpressing Bcl-2exerted almost complete protection from CDDO-Me killing as determined byannexin V-positivity, Phi-Phi-Lux and CMXRos staining. HL-60/Bcl-X_(L)cells were also partially protected from CDDO-Me cytotoxicity (FIG.28A). While Bax protein levels increased in parental HL-60 and inHL-60/Bcl-X_(L) cells, no difference in Bax levels were observed inHL-60/Bcl-2 cells following treatment. In contrast, in p53-negativeHL-60 and HL-60-Dox cells Fas was only not induced but IETD-FMK couldalso not prevent CDDO-Me-induced cell death, indicating involvement ofdifferent mechanisms of apoptotic cell death in these cells (Owen-Schaubet al., 1994). Anti-Fas blocking antibody ZB4 did not preventCDDO-Me-induced killing in NB4 cells favoring direct activation ofcaspase-8. Thus, induction of the FasL/Fas/caspase-8 pathway is notessential in the execution of CDDO-Me-induced cell death.Post-translational modifications of Bcl-2 are involved in the regulationof apoptosis by CDDO-Me. Thus, Bcl-2 phosphorylation is inhibited byCDDO-Me.

[0360] The effect of CDDO-Me on Bcl-2 phosphorylation was furtherinvestigated by performing metabolic labeling studies with³²P-orthophosphoric acid. Following treatment with 0.1 μM of CDDO-Me (aconcentration that induces apoptosis), Bcl-2 phosphorylation wasvirtually abrogated. To further investigate mechanisms of inhibition ofBcl-2 phosphorylation that could play a role in CDDO-Me-induced celldeath, the inventors selected U937 cells that were stably transfectedwith a serine 70→alanine, Bcl-2 mutant (S70A). Previous studiesdemonstrated that the S70A mutant that is unable to be phosphorylated,was also incapable of protecting cells from chemotherapy-inducedapoptosis. The U937/S70A Bcl-2 cells were found to be quite sensitive toCDDO-Me (60% decrease in viability following 0.1 μM of CDDO-Me). Incontrast, a Ser→Glu mutant of Bcl-2, S70E, which may mimic a phosphatecharge and was shown to be able to potently suppress apoptosis, wasfound to be much more resistant to CDDO-Me-induced apoptosis as comparedto wild-type Bcl-2 (75% viable cells after 0.1 μM of CDDO-Me as comparedto 30% for U937/wt). These data indicate that the phosphorylation statusof Bcl-2 contributes to cell sensitivity to CDDO-Me-induced killing. Ofnote, U937/wt and the S70E and S70A Bcl-2 transfectants express roughlyequivalent levels of Bcl2 protein as determined by densitometry,suggesting that the observed differential effects are not related todifferences in Bcl-2 expression.

[0361] Since PKC-α and the MAP kinases ERK1 (p44) and ERK2 (p42) havebeen identified as physiologic Bcl-2 kinases, studies were performed toassess their activation status. Western blot analysis of subcellularfractions of U937 cells revealed that little, if any, PKC-α wasco-localized with Bcl-2 in the mitochondrial membranes of U937 cells,suggesting that mitochondrial PKC is not a likely target of CDDO-Me.However, both ERK1 and ERK2 were prominently detected in themitochondrial membranes. Furthermore, although treatment of cells with 1μM CDDO-Me for 3 hours had no effect on the total ERK1/2 protein levels,CDDO-Me blocked the activation of ERK1/2, as shown by inhibition ofERK1/2 phosphorylation. Treatment of K562 cells with 1 μM of CDDO-Me invivo abrogated ERK kinase activity similar to specific MEK inhibitorPD58059. No effect on Akt activity was seen. These findings indicatethat CDDO-Me induces apoptosis by inhibition of ERK1/2 and Bcl-2phosphorylation.

[0362] To determine specific effects of CDDO-Me on ERK activity theinventors used an in vitro MAPK kinase assay kit. ERK wasimmunoprecipitated from K562 cells since these cells contain the Bcr-Ablkinase and activated ERK present in these cells under basal conditions.CDDO-Me inhibited MBP phosphorylation in a dose-dependent manner.Similar effects were observed in HL-60 cells. These data indicate thatthere may be a direct effect of the compound on ERK kinase activity.

[0363] CDDO-Compounds and Retinoids Synergistically Decrease Viabilityand Induce Differentiation in Leukemic Cell Lines:

[0364] Combinations of CDDO-compounds and retionic acids were tested foranti-cancer therapy.

[0365] CDDO. HL-60 cells in the exponential growth phase were treatedwith 0.1 and 1 μM of CDDO-compounds alone or in combination with 0.5 or1 μM of all-trans-retionic-acid (ATRA). Cell viability was measuredafter 48 hrs using the Cell Titer 96 AQ Non-Radioactive CellProliferation Assay (Promega, Madison, Wis.). Combination of CDDO withATRA significantly decreased the viability of HL-60 cells (FIG. 14).

[0366] Effect of CDDO/ATRA combinations were also tested in differentleukemic cell lines. Leukemic cells cultured at 0.5×10⁶ cells/ml weretreated with CDDO in indicated concentrations, alone or in combinationwith ATRA (FIG. 15). Combination of both agents decreased viable cellnumbers in all cell lines tested by amounts that exceeded the effectsseen by CDDO or ATRA alone.

[0367] Enhancement in cellular differentiation using combinations ofCDDO with ATRA were tested. HL-60 cells were cultured with differentconcentrations of CDDO (0.01, 0.1 and 1 μM) for 72 hrs, alone or incombination with 1 μM ATRA. Cell differentiation was analyzed by flowcytometry (CD14/CD11b staining). CDDO exhibited profound synergism withATRA in induction of monocytic differentiation as demonstrated by CD14induction (FIG. 16). These experiments were repeated 3 times and yieldedessentially identical results.

[0368] The present inventors have previously demonstrated that ATRAdownregulates Bcl-2 mRNA and protein. Therefore, decreases in Bcl-2 mRNAand protein levels were analyzed in cells treated with combinations ofCDDO and ATRA. Combined treatment of U937 cells with CDDO and ATRAinduce significant decrease in Bcl-2 mRNA at 24 hours (FIG. 17). Thesedata were confirmed by quantitative flow cytometry demonstratingdecrease of Bcl-2 protein at 72 hours.

[0369] PPARγ and RXR are known to function as heterodimers. Therefore,the inventors contemplate combination treatments with CDDO-compounds andRXR-specific ligands and/or PPARγ ligands. The RXR-specific ligandLG-100268 (Ligand Pharmaceuticals) used at 1 and 10 nM significantlyenhanced differentiation and cell killing in HL-60 cells (FIG. 18A,Table 5). This induction of differentiation and cell killing was morepronounced compared with ATRA when used at low concentrations (Table 5).TABLE 5 RXR-specific ligand LG-100268 enhances CDDO-induced monocyticdifferentiation in HL-60 cells. CDDO + ATRA CDDO + LG100268 CDDO μM 00.5 1 0 μM 0.5 μM 1 μM No ligand 8.5 36.7 80.4 8.5 36.7 80.4  1 nM 4 5985.6 16.6 91.2 91  10 nM 5.2 70.8 86.5 9.6 83.3 Dead 100 nM 4.4 78.4Dead 9.4 86.6 Dead

[0370] Results are expressed as a percentage of CD14 (+) live cells(FCM).

[0371] To confirm the specificity of the observed effects, HL-60 cellsharboring a dominant-negative mutation in the retinoid receptor, withthe normal retinoid receptors RXR-α and RAR-α was introduced byretroviral gene transfer (HL-60/RXR and HL-60/RAR cell lines. Asdemonstrated in FIG. 18B, RXR-specific ligand LG-100268 increasedCDDO-induced apoptosis only in cells expressing RXR but not RARreceptor.

[0372] CDDO-Me. CDDO-Me and retinoids synergistically decrease viabilityand induce differentiation in leukemic cell lines. The inventors testedthe Effect of CDDO-Me/ATRA combinations in different leukemic celllines. The combination of CDDO-Me with ATRA significantly decreased theviability of HL-60 cells (FIG. 30A) and enhanced apoptosis in U937 cells(FIG. 30B). Combinations of both agents decreased viable cell numbers inall cell lines tested and exceeded the effects seen by CDDO-Me or ATRAalone (FIG. 31) indicating that combinations of both compounds activateapoptotic pathways. This was confirmed in HL-60 cells by annexin Vstaining (17.9 vs 30% positive cells when 0.1 μM of CDDO-Me was combinedwith 1 μM ATRA). In primary AML, ATRA enhanced CDDO-Me-induced apoptosisin {fraction (3/8)} samples tested (an example of subG₁ flow cytometryin AML sample shown in FIG. 32).

[0373] The inventors and others previously demonstrated that ATRAdownregulates Bcl-2 mRNA and protein (Andreeff et al., 1999; Bradbury etal., 1996). Therefore, the effects of combinations of CDDO-Me and ATRAon Bcl-2 protein level were studied. ATRA alone decreased Bcl-2 protein;however, no additive effect on Bcl-2 expression was noted when ATRA wascombined with CDDO-Me.

[0374] Finally, the inventors examined if CDDO-Me combined with ATRAwould enhance differentiation. HL-60 cells were cultured with of 0.1 μMof CDDO-Me for 72 hrs, alone or in combination with 1 μM ATRA. CDDO-Meexhibited profound synergism with ATRA in inducing granulo-monocyticdifferentiation as demonstrated by CD11b induction. At 48 hrs, 67.4% inCDDO-Me-treated cells and 63.5% in ATRA-treated cells were CD11b+(compared with 40% in DMSO controls), while 85% of cells were positivewhen both compounds were given simultaneously. These studies wererepeated 3 times and yielded essentially identical results. Next,combined treatment with CDDO-Me and RXR-specific ligand were tested forpotentiation of anti-leukemic effects. RXR-specific ligand LG-100268(Ligand Pharmaceuticals) used at 10, 100 nm and 1 μM enhanced killing ofHL-60 cells in a dose-dependent manner (Table 6). At 1 μM of LG-100268marked inhibition of cell growth was observed, and the percentage ofcells in S+G₂M decreased by 50%. Collectively, these data demonstratethat CDDO-Me/retinoid combinations markedly decrease cell viability andinduce terminal differentiation in myeloid leukemic cell lines. TABLE 6RXR-specific ligand LG-100268 enhances CDDO-Me-induced monocyticdifferentiation in HL-60 cells (24 hrs). CDDO-Me plus LG-100268 CDDO-MeμM 0 0.1 No Ligand 4.4 22.4  10 nM 6.8 34.4 100 nM 6.2 40.3  1 μM 6.361.6

[0375] CDDO-Compounds Inhibit Formation of Bone Marrow EndothelialStructures:

[0376] CDDO. PPARγ nuclear receptor is expressed in bone marrowendothelial cells, and PPARγ ligands inhibit inhibited endothelial cellproliferation. Recent data demonstrate an important role for theproliferation of endothelial cells, secretion of angiogenic factors anddeveloping angiogenesis in the biology of leukemias. The presentinventors therefore tested the effects of CDDO in endothelial cellassays. The formation of tube-like endothelial structures was performedusing Matrigel®, a basement membrane matrix extracted from theEngelbreth-Holm Swarm mouse sarcoma cell line. Bone marrow endothelialcells with recombinant angiogenic cytokines were incubated in thepresence or absence of CDDO, and formation of tube-like endothelialstructures was assessed by direct observation using a invertedcontrast-phase microscope. CDDO potently inhibited the formation ofcapillary-like structures by bone marrow endothelium (n=3). CDDO alsodiminished the proliferation of cytokine-stimulated vascular endothelialcells (HUVECs) as determined by [³H]-Thymidine incorporation and bydirect observation by inverted contrast-phase microscopy (n=3). Vascularendothelial cells (HUVEC) were grown in EBM-2 or EGM-2 media (Clonetics)containing VEGF, EGF, bFGF and IGF-1. The proliferation of endothelialcells was assessed by inverted contrast-phase microscope after 24 hrs ofCDDO exposure in the indicated concentrations.

[0377] CDDO-Me. The formation of tube-like endothelial structures thatare associated with angiogenesis was analyzed using a Matrigel system(Kubota et al., 1988). Capillary-like structure formation of bone marrowendothelium was inhibited by CDDO-Me. In addition, cytokine-stimulatedvascular endothelial structure formation was also inhibited by CDDO-Meas determined by tritiated thymidine incorporation and phase-contrastmicroscopy. Furthermore, in a murine model of angiogenesis, angiogenicinvasion promoted by ALL BM plasma was abrogated by CDDO-Me at 0.5 μM(n=8). Thus, CDDO-Me also has anti-angiogenic effects.

Example 3 AML NOD/Scid Model

[0378] AML CD34+38-cells are able to repopulate NOD/Scid mice (so calledScid-repopulating cells) (Lapidot et al., 1994). Recent data (Ailles etal., 1999) demonstrated consistent engraftment of AML in NOD/Scid mice:8 weeks after the intravenous injection of 10⁷ AML cells, the averagepercentage of human cells in mouse marrow was 13.3% (5.7% for “good” and20.5% for “poor” cytogenetic abnormalities). These results provide abasis to use AML NOD/Scid system as the best pre-clinical model for AML.The present inventors therefore, established an AML-NOD/Scid model.

[0379] Each mouse was injected with 10⁷ MACS-separated CD34+ leukemiccells. The engraftment of human leukemic cells is determined at 6-8weeks after transplantation by CD45 flow cytometry and Southern blotanalysis using human a-satellite probe for chromosome 17. The clonalityof leukemic cells is determined by FISH based on the known karyotype ofthe samples studied. Under these conditions, consistent engraftment ofAML was performed in 70% of cases. Phenotype of the leukemic cells andcytogenetic profile was similar to the characteristics of the patient'sprimary blasts. The CDDO-compounds are manufactured under GLP conditionsutilizing the RAID program of CTEP as described above. Over 20 grams ofeach CDDO-compound is available for the proposed in vitro and in vivostudies. CDDO-compounds treatments provided to three AML-NOD/Scid miceresulted in lack of leukemic cells in the bone marrow in comparison to 2untreated controls (FIG. 19).

[0380] The effect of CDDO on engraftment of leukemic cells was furthertested in NOD/Scid mice transplanted with 1×10⁶ human leukemic KBM-3cells. Eleven mice were treated with CDDO at 6 mg/kg/day IP (divided in3 injections per day) for 10 days; a control set of 9 mice receivedvehicle alone. The engraftment of human leukemic cells was determined byFISH based on the known karyotype of the cells (trisomy 8) at 5 weeksfollowing transplantation. While 10.6±2.7% cells were leukemic in thebone marrow of the control group (range 0.12-21%), only 3±2.4% leukemiccells were found in CDDO-treated mice (range 0.04-12.5%, p=0.016).{fraction (5/11)} CDDO-treated animals but only {fraction (1/8)}controls had <1% leukemic cells by FISH analysis. Though preliminary,these results indicate anti-leukemic activity of CDDO in vivo.

[0381] CDDO-compounds Induce Growth Inhibition by Induction of Apoptosisand Differentiation in Myeloid Leukemias. Different in vitro and in vivoassays are contemplated to test cell killing induced in stromalcell-supported cultures, in clonogenic assays and in the NOD/Scid modeltransplanted with human leukemic cells. The enhancement ofchemotherapy-induced apoptosis in AML by CDDO-compounds can also beexamined by a person skilled in the art.

[0382] Effect of CDDO-compounds on the Proliferation, Differentiationand Apoptosis of Leukemic Cells from Primary AML. As CDDO-compoundsdecrease proliferation and induce apoptosis and differentiation inleukemic cell lines and in primary AML samples, effects ofCDDO-compounds on allogenic stromal cell layers which resemble the invivo stromal microenvironment can be tested. The inventors contemplateperforming the following studies; experiments will be conducted with 50primary AML samples. Samples containing >80% blasts will be used andsamples with lower blast count will be enriched by magnetic separation(Vario-MACS, Miltenyi Biotech) for CD34+cells. This will include AMLsamples of different FAB subtypes and cytogenetic groups, at least 12samples each will be tested for favorable (t(8;21), inv16, t(15;17)),intermediate (diploid) and poor-prognosis (all other abnormalities)cytogenetics.

[0383] Based on the preliminary paired data (mean=13.5%, std dev=14.4%),a sample size of 12 will yield 80% power with a two-sided one-samplet-test on the paired differences at level of significance 0.05 to testthe hypothesis: Ho: difference in mean apoptosis for CDDO-compounds andDMSO is zero. Ha: CDDO-compounds results in 13.5% (absolute) moreapoptosis than DMSO. (Note: a sample size of 15 will yield 85% powerwith Ha: CDDO-compounds results in 12.0% (absolute) more apoptosis thanDMSO, or 91% power with Ha: CDDO-compounds results in 13.5% (absolute)more apoptosis than DMSO).

[0384] CDDO will be used at concentrations of 0.5, 1, 2 and 5 μM andCDDO-Me at 0.1, 0.3 and 0.5 μM for 72 hours. Effects on cells insuspension and adherent cells will be separately analyzed.Anti-proliferative effects of CDDO-compounds will be analyzed by cellcount and histograms using propidium iodide and the analysis will beperformed after gating on CD34⁺ leukemic cells by flow cytometry.Apoptosis will be assayed by caspase cleavage (Phi-Phi-lux), PS/annexinV, and sub-G₁ DNA fragmentation. Induction of differentiation will beanalyzed by flow cytometry of CD34, CD33, CD14, CD13 and CD11b.

[0385] The effect of CDDO-compounds on the clonogenic leukemic cellswill be tested in the CFU-Blast assay, in extension of the results shownin FIG. 7. Fifty-eight AML samples of different FAB and cytogeneticsgroups (good vs. poor at 29 each) will be analyzed. Based on thepreliminary data (mean=58%, std dev=11.2%) a sample size of 29 willallow the estimation of the % reduction in colony formation of AMLprogenitors (without regard to cytogenetics) with a 95% confidenceinterval with a bound on the error of estimation of 5% and coverageprobability 0.90.

[0386] Effect of CDDO-compounds on NOD/Scid-repopulating AML progenitorcells. Each NOD/Scid mouse will be injected with 10⁷ MACS-separatedCD34+ leukemic cells. The engraftment of human leukemic cells will bedetermined at 6-8 weeks after transplantation by CD45 flow cytometry andSouthern blot analysis using human α-satellite probe for chromosome 17.The clonality of leukemic cells will be determined by FISH based on theknown karyotype of the samples studied. The dose and route ofadministration (IV into tail vein, drinking water or via gavage) ofCDDO-compounds will be determined and are described ahead.

[0387] Effects of CDDO-compounds in the NOD/Scid model using fresh orcryopreserved AML cells will be performed. About 50 AML samples will beanalyzed for each experiment. A minimum of 6 mice will be injected with10⁷ leukemic cells each. Three mice will be treated with CDDO-compounds2 weeks after transplantation and 3 mice will remain untreated. As theleukemic cells will have engrafted in NOD/Scid mice by this timeframe,effects on leukemic cell growth will be determined. The inventors willestimate % reduction in NOD/Scid-repopulating AML cells with a 95%confidence interval.

[0388] As demonstrated above CDDO-compounds enhance ara-C killing inprimary AML cells. Combinations of CDDO-compounds with ara-C andDoxorubicin will be tested at their respective IC₅₀ concentrations inprimary AML (n=10) samples (n=10) in colony-forming assay. Based on thepreliminary paired data (mean=11.4%, std dev=16.3%), a sample size of 19will yield 80% power with a two-sided one-sample t-test on the paireddifferences at level of significance 0.05 to test the hypothesis: H_(o):difference in mean cytotoxicity for (CDDO-compounds+ara-C) and ara-Calone is zero. Ha: (CDDO-compounds+ara-C) results in 11.4% (absolute)more cytotoxicity than ara-C alone. (Note: A sample size of 25 willyield 83% power with Ha: (CDDO-compounds+ara-C) results in 10.0%(absolute) more cytotoxicity than ara-C alone, or 91% power with Ha:(CDDO-compounds+ara-C) results in 11.4% (absolute) more cytotoxicitythan ara-C alone.)

[0389] The normality assumption will be tested for the paireddifferences, and if the assumption is violated, an appropriatenon-parametric procedure will be used to test the median difference incytotoxicity between CDDO-compounds+ara-C and ara-C alone.

[0390] Furthermore, the effects of CDDO-compounds on normalhematopoietic progenitor and stem cells will be tested. For theseexperiments, CD34+ MACS-separated bone marrow or apheresis-derived cellswill be used. Toxic effect of CDDO-compounds will be tested on thesenormal progenitors in clonogenic assays and in the NOD/Scid model. Theroute and concentration of CDDO-compounds will be determined asdescribed ahead. The inventors contemplate that these experiments willidentify a “safe” therapeutic concentration range for CDDO-compounds.Based on preliminary data (mean=26.8%, std dev=7.5%) a sample size of 16will allow the estimation of the % reduction in CFU-GM of normal CD34+cells with a 95% confidence interval with a bound on the error ofestimation of 5% and coverage probability 0.90. (Note: A sample size of29 will allow the estimation of the % reduction in colony formation(without regard to cytogenetics) with a 95% confidence interval with abound on the error of estimation of 3.3% and coverage probability 0.90.The sample size of 29 is that determined above for estimating the %reduction in AML progenitors.)

Example 4 Mechanisms of the Effects of CDDO-Compounds on Apoptosis inLeukemic Cells

[0391] The apoptotic pathways activated in response to theCDDO-compounds with regard to expression levels of Bcl-2 and inductionof the CD95/Fas death receptor will be analyzed.

[0392] Intrinsic (mitochondrial) pathway. The inventors also contemplateexamining if pro-caspase-9 is processed in leukemic cells in vitro inresponse to CDDO-compounds, thereby demonstrating the involvement of themitochondriallcytochrome c pathway. In parallel, the inventors alsocontemplate studying drug-induced changes in the mitochondrial membranepotential ΔΨm using the cationic lipophilic fluorochrome CMXRos (Machoet al., 1996) and release of cytochrome c into the cytosol as assessedby subcellular fractionation studies Jurgensmeier et al., 1998;Matsuyama et al., 1998). To discriminate primary and secondary role ofthe mitochondrial damage in CDDO-compound-induced cell death allcaspases will be blocked using the irreversible pan-caspase inhibitorzVAD-fmk. If mitochondrial damage precedes caspase activation and celldeath, a ΔΨm loss and cytochrome c release despite the presence ofzVAD-fmk is expected.

[0393] For these experiments, HL-60 and U937 cells will be treated invitro with 1 μM CDDO or CDDO-Me in the presence or absence of 100 μM ofzVAD-fmk. At 12, 24, 48 and 72 hours thereafter, ΔΨm will be quantitatedby CMXRos fluorescence and endogenous caspase-3-like activity will bemonitored using the cell-permeable fluorigenic substrate PhiPhi-LUX(Zapata et al., 1998.

[0394] As the Bcl-2 family of proteins are central to the regulation ofthe mitochondrial apoptotic pathway. The key function of Bcl-2-likeproteins is to retain cytochrome c inside the mitochondria (Kluck etal., 1997; Yang et al., 1997). As shown herein CDDO-compounds decreaseBcl-2 expression at the mRNA and protein levels. CDDO-compoundscytotoxicity was also shown in Bcl-2-transfected U937 cells (with a3-fold overexpression in the cells utilized). The inventors will furtherexamine if CDDO-compounds can lower Bcl-2 levels below a criticalthreshold which permits apoptosis, even in cells overexpressing Bcl-2.

[0395] U937 and HL-60-transduced cells, selected for high levels ofBcl-2, and their respective vector-control counterparts will be treatedwith 1 μM CDDO or CDDO-Me for 72 hours. Bcl-2 protein levels will bedetermined by quantitative flow cytometry, and mRNA levels by TaqManPCR. If dissipation of ΔΨm will precede Bcl-2 downregulation andapoptotic cell death in CDDO-compounds-treated cells, the inventors willtest if pharmacological inhibitors of PT cyclosporin A and bongkrekicacid will inhibit mitochondrial alterations and apoptosis.

[0396] Extrinsic pathway. Fas/Fas-ligand can be induced by manycytotoxic drugs and is one of the mechanisms by which anticancer drugskill cells (Friesen et al., 1996). Binding of FasL to Fas results information of the Fas death inducing signaling complex (DISC) with theprodomain of caspase-8 (Boldin et al., 1996; Muzio et al., 1996) andapoptosis. A p53-binding sequence was identified in the Fas promoter(Muller et al., 1998). The present inventors will investigate theactivation of the Fas/Fas-ligand pathway in CDDO-compound mediated celldeath in p53-wt cells (NB4).

[0397] The present inventors have demonstrated induction of CD95/Fasreceptor in leukemic NB4 cells and in primary AML samples. The inventorswill further investigate Fas-L expression levels and caspase-8 cleavageafter treatment with CDDO-compounds. These experiments will be performedin NB4 cells treated with different concentrations of CDDO-compounds(0.5, 1 and 2 μM of CDDO) for 48 hours. Time-course experiments willalso be performed in order to determine the induction of Fas/FasL andcaspase-8 cleavage. In these experiments, Fas levels will be determinedby flow cytometry. Fas-L and caspase-8 will be studied by Western blotanalysis. The inventors contemplate that Fas- or Fas-L induction willprecede capase-8 cleavage and apoptosis. Alternatively, in certain cellsCDDO-compounds may directly induce caspase-8 activation without Fas- orFas-L induction as was demonstrated in other cell systems (Wesselborg etal., 1999). Proteolytic processing of caspase-8, as well as downstreamcaspase-3, 6 and 7 will be monitored by immunoblotting. In parallel, theinventors will also assess caspase activity in cell extracts preparedfrom the same cells, using substrates that are relatively specific forcaspase-8 (IEDT-AFC) and downstream effector caspases such as caspase-3and 7 (DEVD-AFC). Caspase-8-like and caspase-3-like protease activitywill be measured by fluorigenic assays using a spectrofluorometric platereader (EL_(X)808, Bio-Tek Instruments, Inc., Winooski, Vt.) in thekinetic mode with excitation and emission wavelengths of 400 and 505 nm,respectively (Deveraux et al., 1997; Leoni et al., 1998). Activity willbe measured by the release of 7-amino-4-trifluoromethyl-coumarin (AFC)from the synthetic peptidyl substrates.

[0398] The role of Fas in CDDO-compounds-induced apoptosis will also beanalyzed by specific blockade of Fas receptor. Leukemic cells will bepre-treated with a Fas-blocking antibody (such as ZB4, 100 ng/ml,Immunotech, Miami, Fla.) for 1 hour prior to the CDDO-compoundtreatment. The endpoint will be the induction of apoptosis (annexin Vand sub-G₁ DNA content). ZB4 blocked CDDO-compound-induced apoptosisindicates that the Fas/Fas-L interaction contributes significantly tothe observed killing by CDDO-compounds. If no protection against killingby the CDDO-compounds is observed, the inventors will test the effect ofa caspase-8 inhibitor (such as IEDT, Calbiochem, San Diego, Calif.).IEDT protection against the CDDO-compounds indicates that theCDDO-compounds activate caspase-8.

[0399] The inventors have demonstrated the expression of downstreaminhibitors of both, intrinsic and extrinsic pathways, in AML cell linesand clinical samples. Specifically, XIAP and survivin, members of theinhibitor-of-apoptosis protein (IAP) family are overexpressed (Tamm etal., 1999; Carter et al., 1999). The inventors observed downregulationof XIAP mRNA and protein by CDDO. Further analysis of 20 primary AMLsamples with high (n=10) and low (n=10) XIAP and survivin levels withregard to their CDDO-compound sensitivity in stromal cell-supportedculture systems are contemplated. This will elucidate the role of IAPsin the sensitivity and resistance to CDDO-compound-induced apoptosis.

Example 5 Promotion of CDDO-Compounds-Induced Cytotoxic Cell Death andDifferentiation in Leukemic Cells by PPARγ

[0400] The present inventors have shown that CDDO-compounds specificallybind and transcativate the nuclear receptor PPARγ. This receptor and itsheterodimeric partner RXR form a DNA-binding complex that regulatestranscription of several target genes (Kliewer et al., 1992; Tontonoz etal., 1994). Ligation of PPARγ was reported to induce cell cycle arrestand differentiation Wu et al., 1996; Tontonoz and Spiegelman, 1994;Brunet al., 1996).

[0401] PPARγ expression in AML, ALL, and CML (Greene et al., 1995) cellsis known but its biological function in hematopoietic cells has not beenwell investigated. A recent report by R. Evans's group demonstrates thatother PPARγ ligands (15d-PGJ2 at 3 μM and BRL49653 at 5 μM) inducemonocytic differentiation of HL-60 cells but do not exhibit killing(Tontonoz et al., 1998). PPARγ agonists decrease the transcriptionalactivity of Bcl-2-luciferase promoter construct (J Reed). The presentinventors will assess the role of this signaling pathway in regard tothe ability of the CDDO-compounds to induce differentiation andapoptosis.

[0402] Role of PPARγ in CDDO-compound-induced differentiation andapoptosis. To determine whether the expression of PPARγ is required forinduction of differentiation and apoptosis by CDDO-compounds theinventors will examine their effect on the HL-60-derived subline CDM-1,which does not express PPARγ Nagy et al., 1995). Lack of induceddifferentiation and apoptosis in these cells by CDDO-compounds willindicate a critical role of PPARγ signaling in CDDO-compound-induceddifferentiation and/or apoptosis. In addition, PPARγ-receptorantagonists will be investigated for their ability to interfere with theactivity of CDDO-compounds in PPARγ-expressing cells.

[0403] For quantitative assessment of differentiation, a morphologicalassessment of cells will be performed (for example by, Wright-Giemsastained cytospin preparations), immunophenotype will be determined(CD11b/CD14 expression) and nitroblue tetrazolium reduction Drach etal., 1993). HL-60 and CDM-1 cells will be treated with differentconcentrations (0.3, 0.5, 1, and 2 μM) of CDDO for 72 hrs and apoptosiswill be determined as described earlier. All experiments will beperformed in triplicates.

[0404] If CDDO-compounds are found to be partially effectiveindependently of PPARγ, the inventors contemplate that signaling throughPPARγ may be complemented by other orphan receptors. In this case, theinventors contemplate investigating binding and transactivation ofCDDO-compounds with other orphan receptors.

[0405] Comparison of CDDO-compounds with other PPARγ ligands. It isknown that 15d-PGJ2 at 3%M and BRL49653 at 5 μM induces monocyticdifferentiation in HL-60 cells but do not cause cell killing. Incontrast, CDDO induced monocytic differentiation at 0.5 to 1.0 μM andapoptosis at 1 μM in HL-60 cells. Therefore, the present inventors willcompare 15d-PGJ2 (Alexis Corporation, San Diego, Calif.), BRL49653,oxidized low-density lipoprotein (OXLDL)(Intracel, Rockville, Md.) (Nagyet al., 1998) and CDDO from 0.1 to 10 μM in HL-60 and primary AML cells.Studies in primary AML (n=10) will be conducted at IC₅₀ concentrationsand at concentrations that maximally induce differentiation in HL-60cells. These experiments will define the ability of CDDO to inducedifferentiation and apoptosis in comparison to other PPARγ ligands.Similar experiments are contemplated with CDDO-Me.

[0406] Expression of PPARγ. The inventors have demonstrated that PPARγis expressed in myeloid leukemic cell lines tested (HL-60, HL-60-DOX,U937, NB4, KG-1) and in {fraction (9/11)} primary AML samples tested(see FIGS. 14, 15). PPARγ expression was also detected in lymphoid cellsJurkat, IM9, SupM2, Raji, SU-DHL and to a lesser extent in Daudi cells(see FIG. 13B). PPARγ protein was not detected in normal CD34+ cells(n=2). Thus, this invention provides that PPARγ is differentiallyexpressed in normal and leukemic progenitor cells.

[0407] The inventors will further analyze PPARγ mRNA and proteinexpression in primary samples with AML and in normal CD34+cells byNorthern blotting using ³²P-labeled cDNA probe (Tontonoz et al., 1995).The inventors will then determine the effects of CDDO-compounds in thesame samples (n=30) and correlate the efficacy of CDDO-compounds withPPARγ expression. Furthermore, the inventors will study primary AMLsamples with deletion of 3p25 (PPARγ mapping site) that do not expressPPARγ. These studies will determine any correlation between PPARγexpression and the efficacy of CDDO-compounds.

[0408] The present inventors have demonstrated that CDDO-compoundsdownregulate mRNA expression of Bcl-2 anti-apoptotic genes. SeveralPPARγ agonists, including prostaglandin J2 and ciglitazone markedlysuppressed the Bcl-2 promoter in Bcl-2 promoter-luciferase reporterassays. To determine whether changes in mRNA levels of Bcl-2 can beascribed, at least in part, to differences in promoter activity inducedby the CDDO-compounds, the inventors will transfect U937 cells (whichexpresses PPARγ) (Greene et al., 1995) with Bcl-2 luciferase reporterconstructs. The inventors will then test the effect of CDDO or CDDO-Meand the other PPARγ agonist troglitazone on the Bcl-2promoter-luciferase activity in transfected U937 cells.

[0409] Luciferase assay. U937 cells (2×10⁷) will be transfected with10-20 μg luciferase reporter DNA by electroporation at 875 V cm⁻¹, 960μF (Bio-Rad Laboratories). After 1 hour recovery, transfected cells intriplicate samples will be treated with 1 μM CDDO or troglitazone.Luciferase activity will be assayed 18-36 h later using theDual-luciferase reporter assay system (Promega) with the pRL-TK vectoras the internal reporter control. Bcl-2 luciferase reporter encompassesthe human 3.7 Kb Bcl-2 promoter region which contains both P1 and P2transcription initiation sites.

[0410] The CDDO-compounds induce differentiation in leukemia cell linesand primary AML samples. In adipose tissue, ligation of PPARγ is knownto induce differentiation of preadipocyte cells that is mediated bytranscriptional activation of adipocyte-specific genes (Kliewer et al.,1992; Tontonoz et al., 1994). Specific hematopoietic gene promoters havenot been tested for PPAR activation. To determine key downstream targetsof PPARγ activation by CDDO-compounds, the inventors will use the AtlascDNA expression array (Clontech) to identify genes whose expression areregulated by PPARγ activation. The inventors have already shown changesin the expression of 24 genes. Thus, new targets regulated byCDDO-compounds in primary AML samples will be identified by the analysisof isolated AML CD34+ cells (n=10). Differences in the expression levels≧2-fold will be considered significant. Selected targets will beverified by RT-PCR, Northern and Western blotting.

[0411] To determine whether CDDO-compounds directly regulatetranscription of the promoter of the target gene(s) identified by arrayanalysis, the inventors will clone the region of the respective genepromoter into a luciferase reporter vector and cotransfect theseconstructs with CMX-mPPARγ expression vectors into CDM-1 cells.Following transfection, the cells will be treated with vehicle or 1 μMof CDDO or CDDO-Me. These experiments will determine if CDDO-compoundsactivate specific target genes directly through PPARγ.

Example 6 Synergistic Interactions between CDDO-Compounds and Retinoidsin AML

[0412] As demonstrated in Example 2, sub-micromolar combinations of theCDDO-compounds and ATRA or the RXR-specific ligand LG-100268 inducestriking differentiation and apoptosis in HL-60 cells (FIG. 18 forCDDO). It is known that PPARγ and RXR form heterodimers, which uponconcomitant ligation of both receptors exhibit maximal transcriptionalactivity (Kliewer et al., 1992; Tontonoz et al., 1994).

[0413] Interactions of retinoids with specific receptors can induce orsuppress transcription of target genes. RAR can bind both ATRA and itsnaturally occurring double-bond isomer, 9-cis retinoid acid (9-cis RA),whereas RXR bind only 9-cis RA (Heyman et al., 1992). Activation ofRAR-α is sufficient to induce differentiation, (Nagy et al., 1995; Mehtaet al., 1996), whereas activation of RXR-α directly induces apoptosisvia down-modulation of Bcl-2 mRNA and protein (Agarwal and Mehta, 1997).The inventors and others have demonstrated that ATRA transcriptionallydownregulates Bcl-2 in AML (Andreeff et al., 1999). In normalhematopoiesis, Bcl-2 levels decrease dramatically during myeloiddifferentiation (Andreeff et al., 1999). This indicates that Bcl-2functions as a downstream regulator of retinoid-induced cell growth anddifferentiation in hematopoietic cells.

[0414] PPARγ must form a heterodimer with RXR to bind DNA and activatetranscription (Nolte et al., 1998). RXR-specific ligands markedly inducethe binding of the co-activator SRC-1 to PPARγ-RXR heterodimers (Westinet al., 1998), and assembly of this complex results in a large increasein transcriptional activity. The finding that SRC and CBP/p300co-activator proteins possess intrinsic histone acetyltransferaseactivity indicate that ligand-mediated receptor transactivation may alsoinvolve targeted histone acetylation. One method to increase the effectsof PPARγ ligands contemplated herein is by combination with ligandsspecific for RXR. Assembly of this complex results in a large increasein transcriptional activity. For example, in RXR-expressing HL-60 cellsthe inventors observed induction of acetylation of histones H3 and H4 byCDDO alone and in combination with RXR-specific ligand LG-100268 asdetermined by Western blot analysis of nuclear histones (see FIG. 34).TSA, the histone deacetylase inhibitor, served as a positive control.Combination of the PPARγ ligand CDDO with TSA synergistically inducedifferentiation in HL-60 cells and the present inventors contemplatethat this is a result of transcriptional activation of target genes viahistone acetylation.

[0415] Thus, combinations of CDDO-compounds and different retinoids willbe tested in primary AML samples for increased spontaneous andchemotherapy-induced apoptosis. Primary samples will be studied fortheir sensitivity to CDDO-compound/retinoid combinations in suspensionand in stromal-based cultures and in NOD/Scid model transplanted withhuman leukemic cells.

[0416] CDDO-compound/ATRA combinations induce apoptosis anddifferentiation in primary AML samples. The effect of CDDO-compounds andATRA on apoptosis and differentiation will be assessed in 20 primary AMLsamples. The effects of CDDO-compounds and ATRA, alone and incombination, on apoptosis and differentiation of leukemic blasts willalso be investigated. Apoptosis will be assessed by caspase cleavage(Phi-Phi-lux), PS/annexin V, and DNA fragmentation assays as describedin the Examples above. Differentiation will be analyzed by expression ofCD34, CD33, CD14, CD13 and CD11b by flow cytometry. The concentrationlevels of CDDO and CDDO-Me that will be tested initially are 0.1 μM, 0.3μM, 0.5 μM, and 1 μM for 72 hours. The concentrations of ATRA that willbe tested are 0 and 1 μM. This gives 4 concentrations of CDDO-compounds,4 concentrations of CDDO-compounds+ATRA, and 1 concentration of ATRAalone for 4+4+1=9 treatment combinations. The controls will comprise agroup with no treatment with either CDDO-compounds or ATRA. Therefore, 5levels of CDDO-compounds (including 0) and 2 levels of ATRA (including0) for a total of 5×2=10 treatment combinations. Thus, a 5×2 factorialexperiment with 3 observations per treatment will be employed, giving atotal of 30 observations. The data will be analyzed with a two-wayanalysis of variance with main effects (CDDO-compounds and ATRA) and aninteraction term.

[0417] The inventors will therefore identify the combination ofCDDO-compounds and ATRA that maximally induce apoptosis and/ordifferentiation. For evaluation of synergistic interactions theinventors will test a range of concentrations of both compounds andutilize the model described by Chou and Talalay, 1984.

[0418] Similar experiments will be performed with CDDO-compounds and theRXR-α specific ligand LG100268 in primary AML.

[0419] Effects of CDDO-compounds/ATRA and CDDO-compounds/LG100268combinations on NOD/Scid-repopulating AML progenitors. The inventorswill also test the effects of CDDO-compounds and ATRA or LG100268, aloneand in combinations at concentrations identified above in the NOD/Scidmodel using fresh or frozen AML CD34+ cells. Ten AML samples will betested for each combination. A minimum of 6 mice will be injected with10⁷ leukemic CD34+ cells each. Three mice each will be treated with CDDOor CDDO-Me, ATRA, LG100268 and combinations 2 weeks after inoculation ofthe leukemic cells as described above in Example 3.

[0420] Mechanisms of CDDO-compounds/retinoid interactions. The inventorscontemplate that the PPARγ agonist, CDDO-compounds, exerts synergisticeffects with retinoids that activate the RXR receptor, due to thecooperative recruitment of coactivator SRC-1 by PPAR-RXR heterodimers. Asynergistic effect observed with high concentrations of ATRA is due toits conversion into 9-cis RA under culture conditions (Heyman et al.,1992). The ability of ATRA to activate RXR in transactivation assaysthat is attributed to the isomerization into 9-cis-RA has beendemonstrated (Mangelsdorf et al., 1990; Agarwal et al., 1996).

[0421] K562 cells will be transduced with RXR or RAR-α Parental K562cells do not express RXR or RAR-α, are completely resistant to ATRA(Robertson et al., 1991) and will serve as controls. To determinewhether RXR is required for CDDO-compound/retinoid effects on apoptosisand differentiation, if RXR-expressing cells respond toCDDO-compound/ATRA or CDDO-compound/LG100268 combinations, RXR iscritical for CDDO-compound/retinoid interactions. If however,CDDO-compound/ATRA is effective in both, K562/RXR and K562/RAR-α cells,RAR-α may contribute to the combined effect of CDDO-compound/ATRA, bydowregulation of Bcl-2 mRNA. In the latter case, the inventors will alsoinvestigate the effect of the RAR-specific ligand TTNPB (Sigma) incombination with CDDO-compounds.

[0422] To test the effects of ATRA and RXR-specific retinoid LG100268combined with CDDO-compounds in K562 parental, K562/RAR and K562/RXRcells, the inventors will use ATRA and LG100268 at 10⁻⁶ to 10⁻⁹ M and0.3 μM CDDO-compounds (a concentration that does not induce cell death),alone and in combination. K562 cells at the starting concentration0.3×10⁶ cells/ml will be cultured for 48, 72 and 96 hours. Apoptosis anddifferentiation will be determined as described above.

[0423] If RXR signaling is involved, the inventors anticipatesynergistic effect of LG/CDDO-compounds combination in K562/RXR but notin parental or K562/RAR cells. This will be confirmed by the ability ofthe specific RXR antagonist (Ligand Pharmaceuticals, San Diego, Calif.)to block this effect.

[0424] If ATRA at low concentrations is synergistic with CDDO-compoundsin K562/RAR cells, the involvement of RAR receptor signaling will betested. First, a specific activator of RARs TTNPB,((E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid), (Sigma) will be tested at 10⁻⁸ M, in combination with 0.3μM CDDO or CDDO-Me. The pan-RAR-selective analog TTNPB exhibits highaffinity to all three isoforms of RARs and is a potent inducer of theirtranscativation activity. It neither binds to RXR receptors nortransactivates their target gene expression (Nagy et al., 1995). Ifactivation of RARs is involved, combined treatment with CDDO or CDDO-Mewill elicit synergistic responses similar to ATRA in K562/RAR but not inparental or K562/RXR cells. This would be again confirmed by blocking ofRAR-α with the antagonist (Allergan, Irvine, Calif.).K562/RAR-α-transfected cells will be cultured simultaneously with asub-optimal concentration of ATRA (1 and 10 nmol/L) and increasingconcentrations of the RAR-α-antagonist (0.05, 0.5, 1 μM) in the presenceof 0.3 μM of CDDO or CDDO-Me for 72 and 96 hours.

[0425] The inventors have demonstrated that CDDO-compounds combined with1 μM ATRA induce decrease in Bcl-2 mRNA in HL-60 cells. Furtherinvestigation to test if selective RAR/RXR ligands combined withCDDO-compounds will affect Bcl-2 mRNA and protein expression atsub-micromolar concentration are contemplated.

[0426] CDDO-compound/retinoid combinations exert their synergism bycooperative recruitment of co-activator SRC-1 to PPAR-RXR heterodimers.Transcriptional activation by nuclear receptors such as PPARγ andretinoid receptors requires recruitment of co-activator proteins,including SRC-1 (Onate et al., 1995; Kamei et al., 1996; Chakravarti etal., 1996; Torchia et al., 1997; Liu et al., 1998). The inventors willtherefore study the combined effects of RAR-/RXR-specific ligands andCDDO-compounds on SRC-1 interactions with PPARγ-RXR heterodimers. Theability of LG-100268 and TTNPB at 10 nM and at their saturatingconcentration 1 μM, alone and combined with 0.1 and 1 μM of CDDO orCDDO-Me, to recruit ³²P-labelled SRC-163373 to PPAR-RXR heterodimersbound to a PPAR responsive element (Kurokawa et al., 1994) will beanalyzed. It is contemplated that LG-100268 but not TTNPB will inducebinding of SRC-1 to PPARγ-RXR heterodimers, and that it will actsynergistically with PPARγ-ligand CDDO-compounds.

[0427] Identification of new target genes for CDDO-compounds/RXR-ligandcombination. Target genes that are activated by both, CDDO-compounds andthe RXR-ligand LG100268, will be identified. A cell bank consisting of12,000 vials of frozen cells from 2200 patients to will be used toidentify activated genes utilizing the Atlas array (described in theExamples above). cDNA from specific AML samples that show synergisticresponses to CDDO-compounds/retinoids will be analyzed and activatedtarget genes will be validated as described above.

Example 7 PPARγ Nuclear Receptor as a Novel Therapeutic Target

[0428] PPAR-signaling is involved in cancer. The inventors have shownthat the PPARγ protein is expressed in myeloid cell lines and in primaryAML, ALL and CLL samples. CDDO is a PPARγ ligand and binds andtransactivated PPARγ. Effects of the PPARγ ligands such as15-deoxyΔ^(12,14)PGJ2, linoleic acid, thiazolidinediones (TZDs) such astroglitazone, BRL49653, and pioglitazone, L-805645, and GW347845X weretested on the proliferation, apoptosis and differentiation of leukemiccell lines. 15-deoxyΔ^(12,14)PGJ2, TZD and BRL49653 decreased theproliferation of leukemic cells as determined by cell count and ³Hincorporation, with 15-deoxyΔ^(12,14)PGJ2 being most potent (IC50=5-10μM). Six day treatment with 15-deoxyΔ^(12,14)PGJ2, BRL49653 andGW347845X induced CD11b expression in 1L-60 cells. Combination of TGZwith ATRA in U937 and THP1 cells, or 15-deoxyΔ^(12,14)pGJ2 with ATRA inHL-60 cells induced marked myelomonocytic differentiation followed byapoptosis of differentiated cells. TGZ+ATRA synergistically reduced thecolony forming ability of THP1 and U937 cells and induced phagocyticactivity in these cells. CDDO-compounds alone and in combination withretinoids such as ATRA also exert antiproliferative and apoptoticresults on leukemic cell lines and primary AML, CLL and ALL samples invitro as well as decreased Bcl-2 expression in leukemic blasts. Thus,novel PPARγ ligands, alone as well as in combination with retinoids orother chemotherapeutic compounds provide novel therapy for cancersespecially leukemias. Ligation of PPARγ in combination with otherchemotherapeutics especially retinoids provides maximal increase oftranscriptional activity in target genes that control apoptosis anddifferentiation.

Example 8 Toxicity Pharmacokinetics and Tissue Distribution

[0429] Toxicity studies of CDDO-compounds in rats and mice wereperformed. Concentrations that were shown to exert biological effects invitro (600 μg by gavage) did not result in any observed organ toxicityor premature death. Systematic subacute and chronic toxicity studies inBALB/c mice for CDDO-compounds are also presented. An HPLC assay forCDDO and CDDO-Me has been established and validated and will be utilizeto investigate the pharmacokinetics and tissue distribution of the drug.

[0430] Subacute toxicity studies. An important aspect of this inventionof CDDO-compounds and its combination therapies with otherchemotherapeutics is to assess the toxicity and relative therapeuticefficacy of CDDO-compounds. The preliminary toxicity (i.e. determinationof LD50 and MTD [maximum tolerated dose]) of CDDO-compounds was studiedin healthy BALB/C mice (male and female, 25-30 g) after single i. v. ororal injections. Seven different dose levels (10 mice/dose level) wereused after the appropriate pilot dosing experiments to define a MTD dosefor both the oral and iv routes of administration. Animals were observedand weighed daily. The experiment was terminated on day 14. Survivinganimals were sacrificed by exposure to carbon dioxide. The unit-doseeffect line will be constructed and used to calculate lethal doses (LD₁₀and LD₅₀).

[0431] Determination of MTD. 60 female Balb/C mice divided into 12groups of 5 mice. With 5 mice per dose level, six dose levels for IV ororal drug administration were used.

[0432] Intravenous CDDO administration. Groups of mice received CDDOintravenously as a single bolus injection into the tail vein on Day 1.Euthanasia in a closed C0₂ chamber was after 14 days. Mice lost at othertimes are specified. The typical organs from each mouse that areexamined include brain, heart, lungs, spleen, pancreas, kidneys, liver,gastrointestinal tract, lymph nodes, muscle, bone marrow, and skin. Thelesions are described and the diagnoses are listed below for eachanimal. The major findings are given in Table 7 that follows tofacilitate comparing groups of animals. TABLE 7 IV CDDO Administration #Ear Tag Accession Treatment Diagnoses/observations  1. 349 0136  0.3mg/kg 1. Myeloid hyperplasia bone marrow 2. Hyperplasia mesenteric lymphnode  2. 350 0137  0.3 mg/kg 1. Dystrophic calcification epicardium,heart 2. Myeloid hyperplasia bone marrow  3. 351 0138  0.3 mg/kg  Myeloid hyperplasia bone marrow  4. 352 0139  0.3 mg/kg   Myeloidhyperplasia bone marrow  5. 353 0140  0.3 mg/kg 1. Dystrophiccalcification epicardium, heart 2. Myeloid hyperplasia bone marrow 3.Hyperplasia cervical & mesenteric lymph nodes  6. 354 0141  1.0 mg/kg  No significant lesions  7. 355 0142  1.0 mg/kg   Hyperplasia cervicallymph nodes  8. 356 0143  1.0 mg/kg 1. Lymphocytic sialoadenitis 2.Myeloid hyperplasia, bone marrow  9. 357 0144  1.0 mg/kg   Myeloidhyperplasia, bone marrow 10. 358 0145  1.0 mg/kg   Dystrophiccalcification epicardium, heart 11. 359 0146  3.0 mg/kg   Myeloidhyperplasia, bone marrow 12. 360 0147  3.0 mg/kg   Myeloid hyperplasia,bone marrow 13. 361 0148  3.0 mg/kg   Myeloid hyperplasia, bone marrow14. 362 0149  3.0 mg/kg 1. Dystrophic calcification epicardium, heart 2.Myeloid hyperplasia bone marrow 15. 363 0150  3.0 mg/kg   Myeloidhyperplasia, bone marrow 16. 364 0151  10.0 mg/kg 1. Myeloid hyperplasiabone marrow 2. Hyperplasia cervical lymph node 17. 365 0152  10.0 mg/kg  Myeloid hyperplasia, bone marrow 18. 366 0153  10.0 mg/kg 1.Dystrophic calcification epicardium, heart 2. Myeloid hyperplasia spleen& bone marrow 19. 367 0154  10.0 mg/kg 1. Myeloid hyperplasia bonemarrow 2. Hyperplasia cervical lymph node 20. 368 0155  10.0 mg/kg   Nosignificant lesions 21. 369 0156  30.0 mg/kg 1. Myeloid hyperplasia bonemarrow 2. Hyperplasia cervical lymph node 22. 370 0157  30.0 mg/kg 1.Dystrophic calcification epicardium, heart 2. Myeloid hyperplasia bonemarrow 3. Hyperplasia cervical lymph node 23. 371 0158  30.0 mg/kg  Myeloid hyperplasia, bone marrow 24. 372 0159  30.0 mg/kg   Myeloidhyperplasia, bone marrow 25. 373 0160  30.0 mg/kg   Lymphoidhyperplasia, spleen 26. 374 0130 100.0 mg/kg   EUTHANASIA @ 8 hours:moribund 1. Lymphocyte apoptosis thymus, spleen, lymph nodes 2.Dystrophic calcification epicardium, heart 27. 378 0131 100.0 mg/kg  EUTHANASIA @ 9 hours: moribund 1. Lymphocyte apoptosis spleen & lymphnodes 2. Hyperplasia mesenteric lymph nodes 28. 376 0132 100.0 mg/kg  EUTHANASIA @ 9 hours: moribund 1. Lymphocyte apoptosis spleen & lymphnodes 2. Myeloid hyperplasia, bone marrow 29. 375 0134 100.0 mg/kg  EUTHANASIA @ 10 days: tail necrosis 1. Myeloid hyperplasia spleen &bone marrow 2. Hyperplasia cervical lymph node 3. Hyperplasia GALT 30.377 0135 100.0 mg/kg   EUTHANASIA @ 10 days: tail necrosis 1. Myeloidhyperplasia spleen & bone marrow 2. Hyperplasia cervical lymph node

[0433] Oral CDDO administration. Groups of mice received CDDO oralgavage on day 1. Euthanasia was performed in a closed CO₂ chamber after14 days. The typical organs from each mouse that are examined includebrain, heart, lungs, spleen, pancreas, kidneys, liver, gastrointestinaltract, lymph nodes, muscle, bone marrow, and skin. The lesions aredescribed and the diagnoses are listed below for each animal. The majorfindings are given in Table 8 that follows to facilitate comparinggroups of animals. TABLE 8 Oral CDDO Administration # Ear Tag AccessionTreatment Diagnoses/observations  1. 379 0193  0.3 mg/kg 1. Dystrophiccalcification epicardium, heart 2. Myeloid hyperplasia bone marrow 3.Lymphoid hyperplasia spleen  2. 380 0194  0.3 mg/kg 1. Myeloidhyperplasia bone marrow 2. Hyperplasia cervical & mesenteric lymph node 3. 381 0195  0.3 mg/kg 1. Dystrophic calcification epicardium, heart 2.Myeloid hyperplasia bone marrow  4. 382 0196  0.3 mg/kg   No significantlesions  5. 383 0197  0.3 mg/kg 1. Dystrophic calcification epicardium,heart 2. Myeloid hyperplasia bone marrow 3. Lymphoid hyperplasia spleen4. Hyperplasia GALT  6. 384 0198  1.0 mg/kg 1. Dystrophic calcificationepicardium, heart 2. Hyperplasia cervical & mesenteric lymph node  7.385 0199  1.0 mg/kg   Hyperplasia mesenteric lymph node  8. 386 0200 1.0 mg/kg 1. Myeloid hyperplasia bone marrow 2. Hyperplasia cervicallymph node 3. Hyperplasia GALT  9. 387 0201  1.0 mg/kg 1. Dystrophiccalcification epicardium, heart 2. Hyperplasia cervical lymph node 10.388 0202  1.0 mg/kg   No significant lesions 11. 389 0203  3.0 mg/kg  Myeloid hyperplasia bone marrow 12. 390 0204  3.0 mg/kg 1. Dystrophiccalcification epicardium, heart 2. Hyperplasia cervical & mesentericlymph node 13. 391 0205  3.0 mg/kg 1. Myeloid hyperplasia bone marrow 2.Hyperplasia mesenteric lymph node 14. 392 0206  3.0 mg/kg 1. Myeloidhyperplasia bone marrow 2. Hyperplasia cervical lymph node 15. 393 0207 3.0 mg/kg 1. Dystrophic calcification epicardium, heart 2. Myeloidhyperplasia bone marrow 3. Hyperplasia GALT 16. 394 0208  10.0 mg/kg  Myeloid hyperplasia bone marrow 17. 395 0209  10.0 mg/kg 1. Myeloidhyperplasia bone marrow 2. Hyperplasia cervical lymph node 18. 396 0210 10.0 mg/kg 1. Dystrophic calcification epicardium, heart 2. Myeloidhyperplasia bone marrow 19. 397 0211  10.0 mg/kg 1. Chronic-activecystitis & pylonephritis 2. Lymphoid hyperplasia spleen 3. Myeloidhyperplasia bone marrow 20. 398 0212  10.0 mg/kg   No significantlesions 21. 399 0213  30.0 mg/kg 1. Dystrophic calcification epicardium,heart 2. Myeloid hyperplasia bone marrow 3. Hyperplasia cervical &mesenteric lymph node 22. 400 0214  30.0 mg/kg   No significant lesions23. 401 0215  30.0 mg/kg 1. Myeloid hyperplasia bone marrow 2.Hyperplasia GALT 24. 402 0216  30.0 mg/kg 1. Dystrophic calcificationepicardium, heart 2. Hyperplasia cervical lymph node 25. 403 0217  30.0mg/kg   Hyperplasia cervical lymph node 26. 404 0218 100.0 mg/kg   Nosignificant lesions 27. 405 0219 100.0 mg/kg 1. Dystrophic calcificationepicardium, heart 2. Myeloid hyperplasia bone marrow 28. 406 0220 100.0mg/kg   Acute cholecystitis 29. 407 0221 100.0 mg/kg 1. Dystrophiccalcification epicardium, heart 2. Hyperplasia cervical lymph node 30.408 0222 100.0 mg/kg   Myeloid hyperplasia bone marrow

[0434] The majority of animals in both the intravenous and oral groupshave myeloid hyperplasia of the bone marrow. This diagnosis is basedupon finding the medullary cavities of decalcified bones filled withhematopoiesis that shows an overwhelming predominance of maturegranulocytes. A few animals also had myleoid hyperplasia of the red pulpof the spleen (a normal site for extramedullary hematopoiesis). Thesehyperplastic changes are often encountered in mice without an apparenttarget and in the absence of known injury. Myeloid hyperplasia can beverified by doing a differential count on peripheral blood. Ifcirculating granulocytes are not increased, then the appropriatediagnosis for increased granulocytes in the sites of myelopoiesis wouldbe myeloid metaplasia.

[0435] The diagnosis of hyperplasia for lymph nodes, white pulp of thespleen, and gut-associated lymphoid tissue (GALT) is made when follicleswith germinal centers are observed. As with the myeloid hyperplasia,this is a change that is commonly encountered in control, untreated miceand in mice from a wide variety of studies.

[0436] Intravenous CDDO-Me administration. Groups of mice receivedCDDO-Me intravenously as a single bolus injection into the tail vein onDay 1. Euthanasia in a closed CO₂ chamber was after 14 days. Mice lostat other times are specified. The typical organs from each mouse thatare examined include brain, heart, lungs, spleen, pancreas, kidneys,liver, gastrointestinal tract, lymph nodes, muscle, bone marrow, andskin. The lesions are described and the diagnoses are listed below foreach animal. The major findings are given in Table 9 that follows tofacilitate comparing groups of animals. TABLE 9 Intravenous CDDO-MeAdministration # Ear Tag Accession Treatment Diagnoses/Observations  1.431 0270    0 mg/kg 1. Myeloid hyperplasia bone marrow 2. Hyperplasiacervical lymph node  2. 436 0271    0 mg/kg   Myeloid hyperplasia bonemarrow  3. 441 0272    0 mg/kg 1. Dystrophic calcification epicardium,heart 2. Myeloid hyperplasia bone marrow  4. 447 0273    0 mg/kg  Myeloid hyperplasia spleen & bone marrow  5. 452 0274  0.3 mg/kg 1.Myeloid hyperplasia bone marrow 2. Hyperplasia mesenteric & subcutaneouslymph   nodes  6. 428 0275  0.3 mg/kg 1. Dystrophic calcification heartepicardium, heart 2. Myeloid hyperplasia bone marrow  7. 429 0276 mg/kg  Myeloid hyperplasia bone marrow  8. 430 0277  0.3 mg/kg 1. Dystrophiccalcification epicardium, heart 2. Myeloid hyperplasia bone marrow  9.432 0253  1.0 mg/kg   DIED @ 5 minutes 10. 433 0278  1.0 mg/kg  Dystrophic calcification epicardium, heart 11. 434 0279  1.0 mg/kg  Dystrophic calcification epicardium, heart 12. 435 0280  1.0 mg/kg 1.Dystrophic calcification epicardium, heart 2. Myeloid hyperplasia bonemarrow 3. Hyperplasia cervical lymph node 13. 437 0281  3.0 mg/kg  Myeloid hyperplasia bone marrow 14. 438 0282  3.0 mg/kg 1. Myeloidhyperplasia bone marrow 2. Hyperplasia cervical lymph nodes 15. 439 0283 3.0 mg/kg   No significant lesions 16. 440 0284  3.0 mg/kg   Myeloidhyperplasia spleen & bone marrow 17. 443 02825  10.0 mg/kg   Nosignificant lesions 18. 444 0286  10.0 mg/kg   Myeloid hyperplasia bonemarrow 19. 445 0287  10.0 mg/kg   Dystrophic calcification epicardium,heart 20. 446 0288  10.0 mg/kg   Myeloid hyperplasia bone marrow 21. 4480289  30.0 mg/kg   No significant lesions 22. 449 0290  30.0 mg/kg  Myeloid hyperplasia bone marrow 23. 450 0291  30.0 mg/kg   DRUG NOTADMINISTERED 1. Dystrophic calcification epicardium, heart 2.Hyperplasia GALT 24. 451 0292  30.0 mg/kg 1. Myeloid hyperplasia spleen& bone marrow 2. Hyperplasia cervical lymph nodes 25. 453 0293 100.0mg/kg   Myeloid hyperplasia spleen & bone marrow 26. 454 0294 100.0mg/kg 1. Granulosa cell tumor (benign), ovary 2. Dystrophiccalcification epicardium, heart 3. Myeloid hyperplasia spleen & bonemarrow 27. 455 0295 100.0 mg/kg   Myeloid hyperplasia spleen & bonemarrow 28. 456 0296 100.0 mg/kg   Myeloid hyperplasia spleen & bonemarrow

[0437] Oral CDDO-Me administration. Groups of mice received CDDO-Me oralgavage on day 1. Euthanasia was performed in a closed CO₂ chamber after14 days. The typical organs from each mouse that are examined includebrain, heart, lungs, spleen, pancreas, kidneys, liver, gastrointestinaltract, lymph nodes, muscle, bone marrow, and skin. The lesions aredescribed and the diagnoses are listed below for each animal. The majorfindings are given in Table 10 that follows to facilitate comparinggroups of animals. TABLE 10 Oral CDDO-Me Administration # Ear TagAccession Treatment Diagnoses/Observations  1. 501 0304    0 mg/kg  Focal acute to subacute lymphadenitis,   cervical lymph node  2. 5020305    0 mg/kg   No significant lesions  3. 503 0306    0 mg/kg 1.Dystrophic calcification epicardium, heart 2. Hyperplasia cervical lymphnode  4. 504 0307    0 mg/kg   Myeloid hyperplasia bone marrow  5. 5050308    0 mg/kg   Myeloid hyperplasia bone marrow  6. 506 0309  0.3mg/kg 1. Hyperplasia cervical lymph nodes 2. Myeloid hyperplasia bonemarrow  7. 507 0310  0.3 mg/kg   Myeloid hyperplasia bone marrow  8. 0580311  0.3 mg/kg   Myeloid hyperplasia bone marrow  9. 509 0312  0.3mg/kg   Dystrophic calcification epicardium, heart 10. 510 0313  0.3mg/kg 1. Hyperplasia cervical lymph nodes 2. Myeloid hyperplasia bonemarrow 11. 511 0314  1.0 mg/kg   No significant lesions 12. 512 0315 1.0 mg/kg 1. Focal chronic myocarditis, IVS, base of heart 2. Myeloidhyperplasia bone marrow 13. 513 0316  1.0 mg/kg 1. Hyperplasia cervical& mesenteric lymph nodes 2. Chronic, unilateral pyelonephritis 14. 5140317  1.0 mg/kg   No significant lesions 15. 515 0318  1.0 mg/kg  Myeloid hyperplasia bone marrow 16. 516 0319  3.0 mg/kg   Nosignificant lesions 17. 517 0320  3.0 mg/kg 1. Hyperplasia cervicallymph nodes 2. Myeloid hyperplasia bone marrow 18. 518 0321  3.0 mg/kg  Dystrophic calcification epicardium, heart 19. 519 0322  3.0 mg/kg 1.Dystrophic calcification epicardium, heart 2. Myeloid hyperplasia bonemarrow 20. 520 0323  3.0 mg/kg   No significant lesions 21. 521 0324 10.0 mg/kg 1. Dystrophic calcification epicardium, heart 2. Hyperplasiacervical lymph nodes 22. 522 0325  10.0 mg/kg 1. Dystrophiccalcification epicardium, heart 2. Hyperplasia cervical & mesentericlymph nodes 3. Focal granulomatous fasciitis, serosa, urinary bladder23. 523 0326  10.0 mg/kg 1. Hyperplasia cervical lymph nodes 2. Myeloidhyperplasia bone marrow 24. 524 0327  10.0 mg/kg   Dystrophiccalcification epicardium, heart 25. 525 0328  10.0 mg/kg 1. Hyperplasiacervical lymph nodes 2. Subacute, unilateral pyelonephritis 3. Myeloidhyperplasia bone marrow 26. 526 0329  30.0 mg/kg   Dystrophiccalcification epicardium, heart 27. 527 0330  30.0 mg/kg   Dystrophiccalcification epicardium, heart 28. 528 0331  30.0 mg/kg   Dystrophiccalcification epicardium, heart 29. 529 0332  30.0 mg/kg   Nosignificant lesions 30. 530 0333  30.0 mg/kg   No significant lesions31. 531 0334 100.0 mg/kg 1. Necrotizing pericarditis 2. Dystrophiccalcification epicardium, heart 3. Necrotizing pleuritis & pheumonitis4. Myeloid hyperplasia spleen & bone marrow 5. Lymphoid atrophy spleen6. Erythroid atrophy bone marrow 32. 532 0335 100.0 mg/kg   Dystrophiccalcification epicardium, heart 33. 533 0336 100.0 mg/kg   Dystrophiccalcification epicardium, heart 34. 534 0337 100.0 mg/kg   Nosignificant lesions 35. 535 0338 100.0 mg/kg   No significant lesions

[0438] Dystrophic calcification of the epicardium is a common finding inmice (Vargas et al., 1996). The majority of animals in both theintravenous and oral groups have myeloid hyperplasia of the bone marrow.This diagnosis is based upon finding the medullary cavities ofdecalcified bones filled with hematopoiesis that shows an overwhelmingpredominance of mature granulocytes. A few animals also had myleoidhyperplasia of the red pulp of the spleen (a normal site forextramedullary hematopoiesis). These hyperplastic changes are oftenencountered in mice without an apparent target and in the absence ofknown injury.

[0439] The diagnosis of hyperplasia for lymph nodes, white pulp of thespleen, and gut-associated lymphoid tissue (GALT) is made when follicleswith germinal centers are observed. As with the myeloid hyperplasia,this is a change that is commonly encountered in control, untreated miceand in mice from a wide variety of studies.

[0440] Chronic toxicity studies. For these studies, Balb/c mice of bothsexes will be treated with 10 weekly i. v. doses of 20%, 30%, 40% and50% of the predetermined single dose LD10 of CDDO-compounds. Animalswill be weighed weekly and survival rates in different groups will berecorded. Differences in toxic doses between sexes and between routes ofadministration will be determined and compared.

[0441] Analytical Assays and Pharmacokinetics. Understanding thepharmacokinetics and tissue distribution of CDDO-compounds is necessaryfor optimal application of this agent in the clinical setting and foradequate interpretation of preclinical toxicity and efficacy data. It isclear, however, that before pharmacokinetic and biodistribution studiesof CDDO-compounds can proceed, there must be reliable, validated methodswith sufficient sensitivity and specificity to quantify low levels ofCDDO-compounds in relevant biological matrices (i.e. plasma and urine).The inventors have developed and validated an HPLC/UV based method fordetermination of CDDO-compounds. Coordinate with this assay is thedevelopment of a parallel assay using LC/MS that can be used fordetermination of CDDO-compounds metabolites. The drug is well suited toextraction using solid phase C18 cartridges. Analysis of the drug can beaccomplished using one of two methods. The first involves an HPLC(Waters Alliance system) equipped with a Bondapak C18, 5 micron particlesize reverse phase column (with UV detection at 284 nm) where theisocratic mobile phase consists of ammonium acetate buffer andacetonitrile (50:50, v:v). Analyses of the drug and its metabolites canbe determined using a Micromass Platform mass spectrometer withelectrospray ionization (positive mode) where examination of the m/z 492ion [positive electrospray mode] is followed. Both assays use a methylester derivative of CDDO as an internal standard.

[0442] CDDO-compounds prepared in DMSO (10 mg/ml) will be injected i.p.or orally (by gavage) into Balb/c mice at the respective MTD doses. Atselected time points post-injection (5 min, 15, 30, 45 min, 1 hr, 2, 4,8, 12, 24, 48 and 72 hr), groups of mice will be killed (5 mice per timepoint), and plasma prepared from blood. Samples will be frozen at −80°C. until analysis. Separate groups (n 5) of mice will be placed inmetabolism cages and urine and feces collected every 8 hr for 48 hr todetermine elimination of CDDO-compounds and related metabolites. Micesacrificed for pharmacokinetic studies will immediately be used toharvest tissues (e.g. liver, spleen, lung, heart, kidney, smallintestine, large intestine, and skeletal muscle) in order to determinetissue distribution of CDDO-compounds. The 72-hr time point is necessaryto insure adequate sampling of the CDDO-compounds elimination phase.Plasma and urine samples will be extracted and analyzed as previouslydescribed. The concentration of CDDO in each sample will be calculatedby determining the ratio of the CDDO-compounds peak area to that of thecorresponding peak of the internal standard CDDO methyl ester and bycomparing the ratio with a standard curve prepared in the appropriatematrix. All pharmacokinetic parameters will be analyzed bynon-compartmental analysis using the WIN-NONLIN software program.CDDO-compound's elimination half-life (t{fraction (1/2)}), area underthe curve (AUC), volume of distribution (Vd), clearance (Cl), and peakplasma (Cmax) will be calculated. In addition, total fecal and urinaryclearance of CDDO will be determined as well as relative oralbioavailability.

[0443] Tissue distribution. Organs harvested from Balb/c mice at varioustime points post-injection can be harvested, blotted, weighed, andhomogenized. A portion of homogenized samples can be extracted andanalyzed as described above.

[0444] Antitumor Efficacy. The relative antitumor efficacy ofCDDO-compounds and its combinations with chemotherapeutics such asretinoids can also be assessed against both human solid tumors (MX1breast, HT29 colon and BRO melanoma) as well as against human leukemiacell lines in SCID mice.

Example 9 In Vivo Effects of CDDO

[0445] The activity of CDDO against breast cancert cells has also beendemonstrated in vivo. CDDO was given at 20 and 40 mg/kg i.v. twice aweek 10 days after nude mice were injected s.c. with 2×10⁶estrogen-receptor negative, PPARγ positive 435 breast cancer cells.Results shown in FIG. 35 demonstrate that CDDO inhibits the growth ofbreast cancer cells in vivo at both concentrations used.

[0446] All of the compositions and/or methods disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the compositions and/or methods and in the steps or in the sequenceof steps of the method described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

[0447] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference.

[0448] Agarwal and Mehta, “Possible involvement of Bcl-2 pathway inresinoid X receptor alpha-induced apoptosis of HL-60 cells,” BiochemBiophys Res Common, 230(2):25 1-253, 1997.

[0449] Agarwal, Chandraratna, Teng, Nagpal, Rorke, Eckert, “Differentialregulation of human ectocervical epithelial cell line proliferation anddifferentiation by resinoid X receptor- and resinoid acidreceptor-specific retinoids,” Cell Growth Differ, 7(4):521-530, 1996.

[0450] Ailles, Gerhard, Kawagoe, Hogge, “Growth characteristics of acutemyelogenous leukemia progenitors that initiate malignant hematopoiesisin nonobese diabetic/severe combined immunodeficient mice,” Blood,94(5):1761-1772, 1999.

[0451] Andreeff, “Acute myeloid leukemia,” In: Cancer Treatment, Haskell(Ed.), W. B. Saunders, 911-922, 1995.

[0452] Andreeff, Jiang, Zhang, Konopleva, Estrov, Snell, Xie, Okcu,Sanchez-Williams, Dong, Estey, Champion, Komblau, Reed, Zhao,“Expression of bcl-2-related genes in normal and AML progenitors:Changes induced by chemotherapy and cationic acid,” Leukemia,13(11):18811892, 1999.

[0453] Beran, Estey, O'Brian, Cortes, Koller, Giles, Kornblau, Andreeff,Vey, Pierce, Hayes, Wong, Keating, Kantarijian, “Topotecan andcytarabine is an active combination regimen in myelodysplastic syndromesand chronic myelomonocytic leukemia,” J. Clinical Oncology,17(9):2819-2830, 1999.

[0454] Boldin, Goncharov, Goltsev, Wallach, Involvement of MACH, a novelMORT 1/FADD-interacting protease, in Fas/APO-1- and TNF receptorinducedcell death,” Cell, 85(6):803-815, 1996.

[0455] Brun, Tontonoz, Forman, Egis, Chen, Evans, Spiegelman,Differential activation of adipogenesis by multiple PPAR isoforms,”Genes Dev, 10(8):974-984, 1996.

[0456] Carter, Algiers, Andreeff, “Expression of survivin, a member ofthe inhibitor of apoptosis (IAP) family of caspase inhibitors isexpressed in AML and regulated by cytokines and ATRA,” Blood, 94(Sappy1), 1999.

[0457] Castaigne, Chomienne, Daniel, Ballerina, Bergen, Fenaux, Degas,“All-trans resinoid acid as a differentiation therapy for acutepromyelocytic leukemia,” Blood, 76(9):1704-1709, 1990.

[0458] Chakravarti, LaMorte, Nelson, Nakajima, Schulman, Juguilon,Montminy, Evans, “Role of CBP/P300 in nuclear receptor signalling,Nature, 383(659S):99-103, 1996.

[0459] Chou and Talalay, “Quantitative analysis of dose-effectrelationships: the combined effects of multiple drugs or enzymeinhibitors,” Adv Enzyme Regal., 22:27-55:27-55, 1984.

[0460] Deveraux, Roy, Stennicke, Van Arsdale, Zhou, Srinivasula,Alnemri, Salvesen, Reed, “IAPS block apoptotic events induced bycaspase-8 and cytochrome c by direct inhibition of distinct campuses,”EMBO J, 17(8):2215-2223, 1998.

[0461] Deveraux, Takahashi, Salvesen, Reed, “X-linked IAP is a directinhibitor of cell-death pretesses,” Nature, 388(6639): 300-304, 1997.

[0462] Drach, Lopez-Berestein, McQueen, Andreeff, Mehta, “Induction ofdifferation in myeloid leukemia cell lines and acute promyelocyticleukemia cells by liposomal all-trans-retinoic acid,” Cancer Research,53:2100-2102, 1993.

[0463] Estey, Giles, Kantarjian, O'Brien, Cortes, Freireich,LopezBerestein, Keating, “Molecular remissions induced byliposomal-encapsulated all-trans resinoid acid in newly diagnosed acutepromyelocytic leukemia,” Blood, 94:2230-2235, 1999. Pfahl M, Apfel R,Bendik I, Fanjul A, Graupner G, Lee M O, La Vista N, Lu X P,

[0464] Estey, Thall, Pierce, Cortex, Beran, Kantarjian, Keating,Andreeff, Freireich, “Randomized phase II study of fludarabine+cytosinearabinoside+idarubicin+all-trans retinoic acid+granulocyte-colonystimulating factor in poor prognosis newly-diagnosed non-APL, AML andMDS, Blood, 1998.

[0465] Evans, “The steroid and thyroid hormone receptor superfamily,”Science, 240(4854):889-895, 1988.

[0466] Fanjul, Delia, Pierotti, Rideout, Yu, Pfahl, Qiu,“4-Hydroxyphenyl retinamide is a highly selective activator of resinoidreceptors,” J Biol Chem, 271(37):22441-22446, 1996.

[0467] Forman, Chen, Evans, “Hypolipidemic drugs, polyunsaturated fattyacids, and eicosanoids are ligands for peroxisome proliferator-activatedreceptors alpha and delta,” Proc Natl Acad Sci USA, 94(9):4312-43 17,1997.

[0468] Forman, Tontonoz, Chen, Brun, Spiegelman, Evans, 15-Deoxy-delta12, 14-prostaglandin J2 is a ligand for the adipocyte determinationfactor PPAR gamma,” Cell, 83(5):803-812, 1995.

[0469] Friesen, Herr, Krammer, Debatin, “Involvement of the CD95(APO1/FAS) receptor/ligand system in drug-induced apoptosis in leukemiacells,” Nature Med, 2(5):574-577, 1996.

[0470] Friesen, Herr, Krammer, Debatin, “Involvement of the CD95(Apo1/Fas) receptor/ligand system in drug-induced apoptosis in leukemiacells,” Nature Med, 2(5):574-577, 1996.

[0471] Gliniak B, Le T, “Tumor necrosis factor-relatedapoptosis-inducing ligand's antitumor activity in vivo is enhanced bythe chemotherapeutic agent CPT-11”, Cancer Research 59, 6153-6258, 1999.

[0472] Greenberg, Advani, Tallman, Letendre, Saba, Dugan, Lee, Lum,Sikic, Paietta, Bennett, Rowe, “Treatment of refractory/relapsed AMLwith PSC833 plus mitoxantrone, etoposide, cytarabine (PSC-MEC) vs. MEC,randomized phase III trial (E2995),” Blood, 94(1):383a, 1999.

[0473] Greene, Blumberg, McBride, Yi, Kronquist, Kwan, Hsieh, Greene,Nimer, “Isolation of the human peroxisome proliferator activatedreceptor gamma cDNA: expression in hematopoietic cells and chromosomesmapping,” Gene Expr, 4(4-5):281-299, 1995.

[0474] Heyman, Mangelsdorf, Dyck, Stein, Eichele, Evans, Thaller, “9-cisresinoid acid is a high affinity ligand for the resinoid X receptor,”Cell, 63(2):397-406, 1992.

[0475] Jiang, Ting, Seed, “PPAR-gamma agonists inhibit production ofmonocyte inflammatory cytokines,” Nature, 391(6662):82-86, 1998.

[0476] Johansson, Billstrom, Kristoffersson, Akerman, Garmcz, Ahlgren,Maim, Mitelman, “Deletion of chromosome arm 3p in hematologicmalignancies,” Leukemia, 11(8):1207-1213, 1997.

[0477] Jurgensmeier, Xie, Deveraux, Ellerby, Bredesen, Reed, “Baxdirectly induces release of cytochrome c from isolated mitochondria,”Proc Natl Acad Sci USA, 95(9):4997-5002, 1998.

[0478] Keenan, Sato, Marvin, Lander, Gilmour, Mitchell,“IS-Deoxy-Delta(12,14)-prostaglandin J(2), a ligand for peroxisomeproliferator-activated receptor-gamma, induces apoptosis in JEG3choriocarcinoma cells,” Biochem Biophys Res Common, 262(3):579-585,1999.

[0479] Khmer, Xu, Heinzel, Torchia, Kurokawa, Gloss, Lin, Heyman, Rose,Glass, Rosenfeld, “A CBP integrator complex mediates transcription'sactivation and AP-1 inhibition by nuclear receptors,” Cell,85(3):403-414, 1996.

[0480] Kim, Lotan, Yue, Sporn, Suh, Gribble, Honda, Hong, Sun,“Capasase-3 activation is involved in apoptosis induced by a synthetictriterpenoid in Non-small cell lung cancer (NSCLC) cells,” Proc. Amer.Assoc. Cancer Res., 2000

[0481] Kitamura, Miyazaki, Shinomura, Kinds, Kanayama, Matsuzawa,“Peroxisome proliferator-activated receptor gamma induces growth arrestand differentiation markers of human colon cancer cells,” Jpn J CancerRes, 90(1):75-80, 1999.

[0482] Kliewer, Forman, Blumberg, Ong, Borgmeyer, Mangelsdorf, Umesono,Evans, “Differential expression and activation of a family of murineperoxisome proliferator-activated receptors,” Proc Natl Acad Sci USA,91(15):7355-7359, 1994.

[0483] Kliewer, Lenhard, Willson, Patel, Morris, Lehmann, “Aprostaglandin J2 metabolite binds peroxisome proliferator-activatedreceptor gamma and promotes adipocyte differentiation,” Cell, 83(5):813-819, 1995.

[0484] Kliewer, Umesono, Norman, Heyman, Evans, “Convergence of 9-cosresinoid acid and peroxisome proliferator signalling pathways throughheterodimer formation of their receptors,” Nature, 358(63 89):771-774,1992.

[0485] Kluck, Bossy-Wetzel, Green, Newmeyer, “The release of cytochromec from mitochondria: A primary site for bcl-2 regulation of apoptosis,”Science, 275(5303):1132-1136, 1997.

[0486] Kolitz, George, Hurd, Hoke, Dodge, Velez-Garcia, Powell, Moore,Caligiuri, Vardiman, Bloomfield, Larson, “Parallel Phase I trials ofmulti-drug resistance (MDR) modulation with PSC-833 (PSC) in untreatedpatients (pts) with acute myeloid leukemia (AML)<60 years old:preliminary results of CALGB 9621,” Blood, 94(10:1):384a, 1999.

[0487] Konopleva and Andreeff, “Regulatory pathways in programmed celldeath,” Cancer Mol Biol., 6:1229-1260, 1999.

[0488] Konopleva, Estrov, Stiouf, Chang, Zhao, Harris, Leysath, Xie,Jackson, Hong, Honda, Gribble, Place, Suh, Spom, Andreeff, “Novelsynthetic triterpenoid, CDDO, and its methyl ester: Potentantiproliferative, proapoptotic and differentiating agents in AML,”Blood, 94(Sappy 1), 1999.

[0489] Konopleva, Monaco, Zhao, Leysath, Estey, Belmont, Andreeff,“Engraftment potential of AML progenitors into NOD/scid mice isdependent on baseline CXCR4 expression,” Blood, 94(Sappy 1). 1999.

[0490] Konopleva, Zhao, Xie, Segall, Youths, Claxton, Estrov, Komblau,Andreeff, “Apoptosis: molecules and mechanisms,” Adv Exp Med Biol,457:217-236, 1998.

[0491] Kornblau, Konopleva, Andreeff, Apoptosis regulating proteins astargets of therapy for hematological malignancies. Exp. Opin. Inv. Drugs8:2027-2057, 1999.

[0492] Kornblau, Estey, Madden, Tran, Zhao, Consoli, Snell,Sanchez-Williams, Kantarjian, Keating, Newman, Andreeff, “Phase I studyof mitoxantrone plus etoposide with multidrug blockage by SDZ PSC-833 inrelapsed or refractory acute myelogenous leukemia,” J. Clin. Oncol.,15(5):1796-1802, 1997.

[0493] Kurokawa, DiRenzo, Boehm, Sugarman, Gloss, Rosenfeld, Heyman,Glass, “Regulation of resinoid signalling by receptor polarity andallosteric control of ligand binding,” Nature, 371(6497):528-531, 1994.

[0494] Lapidot, Sirard, Vormoor, Murdoch, Hoang, Caceres-Cortes, Minden,Paterson, Caligiuri, Dick, “A cell initiating human acute myeloidleukaemia after transplantation into SCID mice,” Nature,367(6464):645-648, 1994.

[0495] Lehmann, Lenhard, Oliver, Ringold, Kliewer, “Peroxisomeproliferator-activated receptors alpha and gamma are activated byindomethacin and other non-steroidal anti-inflammatory drugs,” J BiolChem, 272(6):3406-3410, 1997.

[0496] Lehmann, Moore, Smith-Oliver, Wilkison, Willson, Kliewer, “Anantidiabetic thiazolidinedione is a high affinity ligand for peroxisomeproliferator-activated receptor gamma (PPAR gamma),” J Biol Chem,270(22): 12953-12956, 1995.

[0497] Leoni, Chao, Cottam, Gemini, Rosenbach, Carrara, Budihardjo,Wang, Carson, “Induction of an apoptotic program in cell-ftee extractsby 2-chloro-2′-deoxyadenosine 5′-triphosphate and cytochrome,” Proc NatlAcad Sci USA, 95(16):9567-9571, 1998.

[0498] Liu, Yao, Kirschenbaum, Levine, “NS398, a selectivecyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2expression in LNCaP cells,” Cancer Res, 58(19):4245-4249, 1998.

[0499] Macho, Decaudin, Castedo, Hirsch, Susin, Zamzami, Kroemer,“Chloromethyl-X-Rosamine is an aldehyde-fixable potential-sensitivefluorochrome for the detection of early apoptosis,” Cytometry,25(4):333-340, 1996.

[0500] Mangelsdorf, Ong, Dyck, Evans, “Nuclear receptor that identifiesa novel resinoid acid response pathway,” Nature, 345(6272):224-229,1990.

[0501] Matsuyama, Xu, Velours, Reed, “The Mitochondrial FOF1-ATPaseproton pump is required for function of the proapoptotic protein Bax inyeast and mammalian cells,” Mol Cell, 1(3):327-336, 1998.

[0502] Mehta, McQueen, Neamati, Collins, Andreeff, “Activation ofresinoid receptors RAR alpha and RXR alpha induces differentiation andapoptosis, respectively, in HL-60 cells,” Cell, Growth Differ, 7(2):179-186, 1996.

[0503] Mueller, Sarraf, Tontonoz, Evans, Martin, Zhang, Fletcher,Singer, Spiegelman, Terminal differentiation of human breast cancerthrough PPAR gamma,” Mol Cell, 1(3):465-470, 1998.

[0504] Mukherjee, Davies, Crumble, Bischoff, Cesario, Jow, Hamann,Boehm, Monday, Nadzan, Patemiti, Heyman, “Sensitization of diabetic andobese mice to insulin by resinoid X receptor agonists,” Nature,386(6623):407-410, 1997.

[0505] Mullet, Wilder, Bannasch, Israeli, Lehibach, Li-Weber, Friedman,Galle, Stremmel, Oren, Krammer, “ps3 activates the CD95 (APO1/Fas) genein response to DNA damage by anticancer drugs,” J Exp Med,188(11):2033-204S, 1998.

[0506] Muzio, Chinnaiyan, Kischkel, O'Rourke, Shevchenko, Ni, Scaffidi,Brett, Zhang, Rentz, Rummer, Peter, Rixit, “FLICE, a novelFADD-homologous ICE/CED-3-like protease, is recruited to the CD95(Fas/APO-1) death-inducing signaling complex,” Cell, 85(6):817-827,1996.

[0507] Nagy, Thomazy, Shipley, Fesus, Lymph, Heyman, Chandraratna,Davies, “Activation of resinoid X receptors induces apoptosis in HL-60cell lines,” Mol Cell Biol, 15(7):3540-3551, 1995.

[0508] Nagy, Thomazy, Shipley, Fesus, Lymph, Heyman, Chandraratna,Davies, “Activation of resinoid X receptors induces apoptosis in HL-60cell lines,” Mol Cell Biol, 15(7):3540-3551, 1995.

[0509] Nagy, Tontonoz, Alvarez, Chen, Evans, “Oxidized LDL regulatesmacrophage gene expression through ligand activation of PPARgamma,”Cell, 93(2):229-240, 1998.

[0510] Nichols, Parks, Consler, Blanchard, “Development of ascintillation proximity assay for peroxisome proliferator-activatedreceptor gamma ligand binding domain,” Anal Biochem, 257(2):112-119,1998.

[0511] Nolte, Wisely, Weston, Cobb, Lambert, Kurokawa, Rosenfeld,Willson, Glass, Milbum, “Ligand binding and co-activator assembly of theperoxisome proliferator-activated receptor-gamma,” Nature, 395(6698):137-143, 1998.

[0512] Onate, Tsai, Tsai, O'Malley, “Sequence and characterization of adeactivator for the steroid hormone receptor superfamily,” Science,270(5240): 1354-1357, 1995.

[0513] Owen-Schaub, Radinsky, Kruzgl, Berry, Yonehara, “Anti-Fas onnonhematopoietic tumors: levels of Fas/APO-1 and bcl-2 are notpredictive of biological responsiveness,” Cancer Res., 54(6):1580-1586,1994.

[0514] Piedrafita and Ortiz, “Nuclear resinoid receptors and theirmechanism of action,” Vitam. Horm., 49:327-82:327-382, 1994.

[0515] Ricote, Li, Willson, Kelly, Glass, “The peroxisomeproliferator-activated receptor-gamma is a negative regulator ofmacrophage activation,” Nature, 391(6662):79-82, 1998.

[0516] Robertson, Mueller, Collins, “Resinoid acid receptors in myeloidleukemia:

[0517] characterization of receptors in resinoid acid-resistant K-562cells,” Blood, 77(2):340-347, 1991.

[0518] Sarraf, Mueller, Smith, Wright, Kum, Aaltonen, de la, Spiegelman,Eng, “Loss-of-function mutations in PPAR gamma associated with humancolon cancer,” Mol Cell, 3(6):799-804, 1999.

[0519] Schadendorf D, Kern M A, Artuc M, Pahl H L, Rosenbach T, FichtnerI, Nurnberg W, Stuting S, von Stebut E, Worm M, Makki A, Jurgovsky K,Kolde G, Henz B M, “Treatment of melanoma cells with the syntheticretinoid CD437 induces apoptosis via activation of AP-1 in vitro, andcauses growth inhibition in xenografts in vivo”,J. Cell Biol,135:1889-1898, 1996.

[0520] Suh, Wang, Honda, Gribble, Dmitrovsky,k Hickey, Maue, Place,Porter, Spinella, Williams, Wu, Dannenberg, Flanders, Letterio,Mangelsdorf, Nathan, Nguyen, Porter, Ren, Roberts, Roche, Subbaramaiah,Sporn, “A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating,antiproliferative, and anti-inflammatory activy,” Cancer Res.,59(2):336-341, 1999.

[0521] Tamm, Segall, Kitada, Scudiero, Tudor, Myers, Monks, Andreeff,Reed, “Expression of IAP-family genes in human cancers and leukemias,”Blood, Suppl. 1, 1999.

[0522] Tontonoz, Hu, Devine, Belle, Spiegelman, “PPAR gamma 2 regulatesadipose expression of the phosphoenolpyruvate carboxykinase gene,” MolCell Biol, 15(1):351-357, 1995.

[0523] Tontonoz, Hu, Graves, Budavari, Spiegelman, “mPPAR gamma 2:tissue-specific regulator of an adipocyte enhancer,” Genes Dev,8(10):12241234, 1994.

[0524] Tontonoz, Hu, Spiegelman, “Stimulation of adipogenesis infibroblasts by PPAR gamma 2, a lipid-activated transcription factor,Cell, 79(7): 1147-1156, 1994.

[0525] Tontonoz, Nagy, Alvarez, Thomazy, Evans, “PPARgamma promotesmonocyte/macrophage differentiation and uptake of oxidized LDL,” Cell,93(2):241-252, 1998.

[0526] Tontonoz, Singer, Forman, Sarraf, Fletcher, Fletcher, Brun,Mueller, Altiok, Oppenheim, Evans Spiegelman, “Terminal differentiationof human liposarcoma cells induced by ligands for peroxisomeproliferator-activated receptor gamma and the resinoid X receptor,” ProcNatl Acad Sci USA, 94(1):237-241, 1997.

[0527] Torchia, Rose, Inostroza, Khmer, Weston, Glass, Rosenfeld, “Thetranscription's coactivator p/CIP binds CBP and mediatesnuclear-receptor function,” Nature, 387(6634):677-684, 1997.

[0528] Vargas et al., “Dystrophic cardiac calcinosis in C3H/HeN mice,”Lab. Anim. Sci. 46:572-575, 1996.

[0529] Veiga, Keenan, Barata, Sallan, Nudger, Cardoso, “Peroxisomeproliferator-activated receptor-gamma (PPAR gamma) expression by bonemarrow endothelium reveals a potential target for therapeuticintervention in acute lymphoblastic leukemia,” Blood, 94:10(1):627a.1999.

[0530] Vermes, Haanen, Stiffens-Nakken, Reutelingsperger, “A novel assayfor apoptosis. Flow cytometric detection of phosphatidylserineexpression on early apoptotic cells using fluorescein labelled AnnexinV,” J Immunol Methods, 184(1):39-51, 1995.

[0531] Verna, Wang, Rao, Tang, Chen, Kramer, Grant, “Induction ofapoptosis and differentiation by fludarabine in human leukemia cells(U937): interactions with the macrocyclic lactose bryostatin 1,”Leukemia, 13(7):10461055, 1999.

[0532] Walczak H, Miller R E, Ariail K, Gliniak B, Griffith T S, KubinM, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin R G,Rauch C T, Schuh J C, Lynch D H, “Tumoricidal activity of tumor necrosisfactor-related apoptosis-inducing ligand in vivo”, Nature Medicine,5:157-163, 1999.

[0533] Wang Y, Porter W W, Suh N, Honda T, Gribble G W, Leesnitzer L M,Plunket K D, Mangelsdorf D J, Blanchard S G, Willson T M, Sporn M B: Asynthetic triterpenoid, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid(CDDO), is a ligand for the peroxisome proliferator-activated receptorgamma. Mol.Endocrinol. 14: 1550-1556, 2000.

[0534] Warrens, Frankel, Wilson, “Differentiation therapy of acutepromyelocytic leukemia with tretinoin (all-trans-retinoic acid),” N EnglJ Med, 324:1385-1393, 1991.

[0535] Wesselborg, Engulf, Rossmann, Los, Schultz-Osthoff, “Anticancerdrugs induce caspase-8/FLICE activation and apoptosis in the absence ofCD9S receptor/ligand interaction,” Blood, 93(9):3053-3063, 1999.

[0536] Westin, Kurokawa, Nolte, Wisely, Mclnerney, Rose, Milbum,Rosenfeld, Glass, “Interactions controlling the assembly ofnuclearreceptor heterodimers and coactivators,” Nature,395(6698):199-202, 1998.

[0537] Willson, Cobb, Cowan, Wiethe, Cornea, Prakash, Beck, Moore,Kliewer, Lehmann, “The structure-activity relationship betweenperoxisome proliferator-activated receptor gamma egotism and theantihyperglycemic activity of thiazolidinediones,” J Med Chem,39(3):665-668, 1996.

[0538] Wu, Bucher, Farmer, “Induction of peroxisomeproliferator-activated receptor gamma during the conversion of 3T3fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta, andglucocorticoids,” Mol Cell Biol, 16(8):4128-4136, 1996.

[0539] Xie, Zhao, Xu, McQueen, Andreeff, “Differential expressionpatterns in human myeloblastic leukemia HL-60 and multidrug resistantHL-60/Dox cells analyzed by human cDNA expression array,” Blood, 92(Sappy 1)[10], 387a. 1998.

[0540] Yang, Liu, Bhalla, Kim, Abrade, Cai, Peng, Jones, Wang,“Prevention of apoptosis by bcl-2: Release of cytochrome c frommitochondria blocked,” Science, 275(S303):1129-1132, 1997.

[0541] Yang, Liu, Bhalla, Kim, Ibrado, Cai, Peng, Jones, Wang,“Prevention of apoptosis by Bcl-2: release of cytochrome c frommitochondria blocked,” Science, 275(5303):1129-1132, 1997.

[0542] Zapata, Takahashi, Salvesen, Reed, “Granzyme release and caspaseactivation in activated human T-lymphocytes,” J Biol Chem,273(12):69166920, 1998.

What is claimed is:
 1. A method for inducing cytotoxicity in a cellcomprising contacting said cell with a CDDO-compound and achemotherapeutic agent, wherein the combination of the CDDO-compoundwith the chemotherapeutic agent is effective in inducing cytotoxicity insaid cell.
 2. The method of claim 1, wherein said CDDO-compound is CDDO.3. The method of claim 1, wherein said CDDO-compound is methyl-CDDO. 4.The method of claim 1, wherein the CDDO-compound is contacted with saidcell prior to contacting said cell with said chemotherapeutic agent. 5.The method of claim 1, wherein said chemotherapeutic agent is contactedwith said cell prior to contacting said cell with CDDO.
 6. The method ofclaim 1, wherein said cell is a cancer cell.
 7. The method of claim 6,wherein said cancer cell is a leukemic cell.
 8. The method of claim 7,wherein said leukemic cell is a blood cancer cell, a myeloid leukemiacell, a monocytic leukemia cell, a myelocytic leukemia cell, apromyelocytic leukemia cell, a myeloblastic leukemia cell, a lymphocyticleukemia cell, an acute myelogenous leukemic cell, a chronic myelogenousleukemic cell, a lymphoblastic leukemia cell, a hairy cell leukemiacell.
 9. The method of claim 6, wherein said cancer cell is a solidtumor cell.
 10. The method of claim 9, wherein said solid tumor cell isa bladder cancer cell, a breast cancer cell, a lung cancer cell, a coloncancer cell, a prostate cancer cell, a liver cancer cell, a pancreaticcancer cell, a stomach cancer cell, a testicular cancer cell, a braincancer cell, an ovarian cancer cell, a lymphatic cancer cell, a skincancer cell, a brain cancer cell, a bone cancer cell, a soft tissuecancer cell.
 11. The method of claim 1, wherein said cell is located ina human subject.
 12. The method of claim 11, wherein said CDDO-compoundis administered locally.
 13. The method of claim 12, wherein saidCDDO-compound is administered by direct intratumoral injection.
 14. Themethod of claim 12, wherein said CDDO-compound is administered byinjection into tumor vasculature.
 15. The method of claim 11, whereinsaid CDDO-compound is administered systemically.
 16. The method of claim15, wherein the CDDO-compound is administered intravenously.
 17. Themethod of claim 15, wherein the CDDO-compound is administeredintra-arterially.
 18. The method of claim 15, wherein the CDDO-compoundis administered intra-peritoneally.
 19. The method of claim 15, whereinthe CDDO-compound is administered orally.
 20. The method of claim 15,wherein the CDDO-compound is administered during ex vivo purging. 21.The method of claim 1, wherein said chemotherapeutic agent isdoxorubicin, decitabine, daunorubicin, dactinomycin, mitoxantrone,cisplatin, procarbazine, mitomycin, carboplatin, bleomycin, etoposide,teniposide, mechlroethamine, cyclophosphamide, ifosfamide, melphalan,chlorambucil, ifosfamide, melphalan, hexamethylmelamine, thiopeta,busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine,adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, hydrogenperoxide, nitrosurea, plicomycin, tamoxifen, taxol, transplatinum,vincristin, vinblastin, TRAIL, dolastatin-10, bryostatin, annamycin,mylotarg, sodium phenylacetate, sodium butyrate, methotrexate, acortocosteroid or tacrolimus.
 22. The method of claim 1, wherein saidchemotherapeutic agent is a retinoid.
 23. The method of claim 22,wherein said retinoid is selected from the group comprisingall-trans-retinoic acid, 9-cis-retinoic acid, LG100268, LGD1069,fenretinide, CD437, a RAR-specific retinoic acid and a RXR-specificretinoic acid.
 24. The method of claim 23, wherein said RXR-specificretinoic acid is LG100268.
 25. The method of claim 1, wherein said cellis contacted with the CDDO-compound a second time.
 26. The method ofclaim 1, wherein said cell is contacted with said chemotherapeutic agenta second time.
 27. The method of claim 1, wherein the CDDO-compound andsaid chemotherapeutic agent are contacted with said cell at the sametime.
 28. The method of claim 11, further comprising tumor resection.29. The method of claim 28, wherein said tumor resection occurs prior tosaid contacting.
 30. The method of claim 28, wherein said contactingcomprises treating a resected tumor bed with the CDDO-compound and saidchemotherapeutic agent.
 31. The method of claim 28, wherein said tumorresection occurs after said contacting.
 32. The method of claim 28,wherein said contacting occurs both before and after said tumorresection.
 33. A method of killing a tumor cell comprising contactingsaid tumor cell with a CDDO-compound and a chemotherapeutic agent,wherein the combination of said CDDO-compound with said chemotherapeuticagent, induces killing of said tumor cell.
 34. The method of claim 33,wherein said CDDO-compound is CDDO.
 35. The method of claim 33, whereinsaid CDDO-compound is methyl-CDDO.
 36. The method of claim 33, whereinsaid chemotherapeutic agent is a retinoid.
 37. A method of inducingapoptosis in a tumor cell comprising contacting said tumor cell with aCDDO-compound and a chemotherapeutic agent, wherein the combination ofsaid CDDO-compound with said chemotherapeutic agent, induces apoptosisof said tumor cell.
 38. The method of claim 37, wherein saidCDDO-compound is CDDO.
 39. The method of claim 37, wherein saidCDDO-compound is methyl-CDDO.
 40. The method of claim 37, wherein saidchemotherapeutic agent is a retinoid.
 41. A method of inducingdifferentiation in a tumor cell comprising contacting said tumor cellwith a CDDO-compound and a chemotherapeutic agent, wherein thecombination of said CDDO-compound with said chemotherapeutic agent,induces the differentiation of said tumor cell.
 42. The method of claim41, wherein said CDDO-compound is CDDO.
 43. The method of claim 41,wherein said CDDO-compound is methyl-CDDO.
 44. The method of claim 41,wherein said chemotherapeutic agent is a retinoid.
 45. A method oftreating cancer in a human patient comprising administering aCDDO-compound and a chemotherapeutic agent to said human patient,wherein the combination of said CDDO-compound with said chemotherapeuticagent, is effective to treat said cancer.
 46. The method of claim 45,wherein said CDDO-compound is CDDO.
 47. The method of claim 45, whereinsaid CDDO-compound is methyl-CDDO.
 48. The method of claim 45, whereinsaid chemotherapeutic agent is a retinoid.
 49. A method of potentiatingthe effect of a chemotherapeutic agent on a tumor cell comprisingcontacting said tumor cell with a CDDO-compound and the chemotherapeuticagent.
 50. The method of claim 49, wherein said CDDO-compound is CDDO.51. The method of claim 49, wherein said CDDO-compound is methyl-CDDO.52. The method of claim 49, wherein said chemotherapeutic agent is aretinoid.
 53. A method of inhibiting growth of a tumor cell comprisingcontacting said tumor cell with a CDDO-compound and a chemotherapeuticagent.
 54. The method of claim 53, wherein said CDDO-compound is CDDO.55. The method of claim 53, wherein said CDDO-compound is methyl-CDDO.56. The method of claim 53, wherein said chemotherapeutic agent is aretinoid.
 57. A method of inducing apoptosis in a lymphoid cell thatexpresses Bcl-2 comprising contacting said lymphoid cell with aCDDO-compound and an immunosupressive agent.
 58. The method of claim 57,wherein the Bcl-2 is endogenous.
 59. The method of claim 57, wherein theBcl-2 is exogenous.
 60. The method of claim 59, wherein the Bcl-2 isexpressed by a expression vector that comprises a nucleic acid thatencodes Bcl-2 under the control of a promoter active in the lymphoidcell.
 61. The method of claim 57, wherein the lymphoid cell is a T-cell.62. The method of claim 57, wherein the lymphoid cell is a cancer cell.63. The method of claim 57, wherein the lymphoid cell is located in ahuman.
 64. The method of claim 57, where the immunosupressive agent is acorticosteroid.
 65. The method of claim 57, where the immunosupressiveagent is a tacrolimus.
 66. The method of claim 57, wherein the lymphoidcell is further contacted with a chemotherapeutic agent.
 67. A method oftreating or preventing graft versus host disease in a subject comprisingadministering to the subject a CDDO-compound in combination with animmunosupressive agent.
 68. The method of claim 67, wherein the subjectis further treated with a chemotherapeutic agent.
 69. The method ofclaim 67, wherein said CDDO-compound is CDDO.
 70. The method of claim67, wherein said CDDO-compound is methyl-CDDO.
 71. The method of claim67, where the immunosupressive agent is a corticosteroid.
 72. The methodof claim 67, where the immunosupressive agent is a tacrolimus.
 73. Themethod of claim 67, where the subject is a human.
 74. The method ofclaim 67, where the subject has cancer.
 75. The method of claim 67,where the subject has received autologus bone marrow transplantation.76. The method of claim 67, wherein the CDDO-compound is administeredduring ex vivo purging.
 77. The method of claim 67, wherein theCDDO-compound is administered locally, by direct intratumoral injectionor by injection into tumor vasculature.
 78. The method of claim 67,wherein said CDDO-compound is administered systemically.
 79. The methodof claim 78, wherein the CDDO-compound is administered intravenously,intra-arterially, intra-peritoneally, or orally.